Publications by authors named "Harini Krishnan"

29 Publications

  • Page 1 of 1

Evidence that Maackia amurensis seed lectin (MASL) exerts pleiotropic actions on oral squamous cells with potential to inhibit SARS-CoV-2 infection and COVID-19 disease progression.

Exp Cell Res 2021 Apr 3;403(1):112594. Epub 2021 Apr 3.

Department of Molecular Biology, And Graduate School of Biomedical Sciences, School of Osteopathic Medicine, Rowan University, Stratford, NJ, 08084, USA. Electronic address:

COVID-19 was declared an international public health emergency in January, and a pandemic in March of 2020. There are over 125 million confirmed COVID-19 cases that have caused over 27 million deaths worldwide as of March 2021. COVID-19 is caused by the SARS-CoV-2 virus. SARS-CoV-2 presents a surface "spike" protein that binds to the ACE2 receptor to infect host cells. In addition to the respiratory tract, SARS-Cov-2 can also infect cells of the oral mucosa, which also express the ACE2 receptor. The spike and ACE2 proteins are highly glycosylated with sialic acid modifications that direct viral-host interactions and infection. Maackia amurensis seed lectin (MASL) has a strong affinity for sialic acid modified proteins and can be used as an antiviral agent. Here, we report that MASL targets the ACE2 receptor, decreases ACE2 expression and glycosylation, suppresses binding of the SARS-CoV-2 spike protein, and decreases expression of inflammatory mediators by oral epithelial cells that cause ARDS in COVID-19 patients. In addition, we report that MASL also inhibits SARS-CoV-2 infection of kidney epithelial cells in culture. This work identifies MASL as an agent with potential to inhibit SARS-CoV-2 infection and COVID-19 related inflammatory syndromes.
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http://dx.doi.org/10.1016/j.yexcr.2021.112594DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8019238PMC
April 2021

Interdomain interactions regulate the localization of a lipid transfer protein at ER-PM contact sites.

Biol Open 2021 Mar 18;10(3). Epub 2021 Mar 18.

Cellular Organization and Signalling, National Centre for Biological Sciences, TIFR-GKVK Campus, Bellary Road, Bengaluru 560065, India

During phospholipase C-β (PLC-β) signalling in photoreceptors, the phosphatidylinositol transfer protein (PITP) RDGB, is required for lipid transfer at endoplasmic reticulum (ER)-plasma membrane (PM) contact sites (MCS). Depletion of RDGB or its mis-localization away from the ER-PM MCS results in multiple defects in photoreceptor function. Previously, the interaction between the FFAT motif of RDGB and the integral ER protein dVAP-A was shown to be essential for accurate localization to ER-PM MCS. Here, we report that the FFAT/dVAP-A interaction alone is insufficient to localize RDGB accurately; this also requires the function of the C-terminal domains, DDHD and LNS2. Mutations in each of these domains results in mis-localization of RDGB leading to loss of function. While the LNS2 domain is necessary, it is not sufficient for the correct localization of RDGB, which also requires the C-terminal DDHD domain. The function of the DDHD domain is mediated through an intramolecular interaction with the LNS2 domain. Thus, interactions between the additional domains in a multi-domain PITP together lead to accurate localization at the MCS and signalling function.This article has an associated First Person interview with the first author of the paper.
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http://dx.doi.org/10.1242/bio.057422DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7990853PMC
March 2021

Evidence that Maackia amurensis seed lectin (MASL) exerts pleiotropic actions on oral squamous cells to inhibit SARS-CoV-2 infection and COVID-19 disease progression.

Res Sq 2020 Oct 23. Epub 2020 Oct 23.

Department of Molecular Biology, and Graduate School of Biomedical Sciences, School of Osteopathic Medicine, Rowan University, Stratford, NJ 08084, USA.

COVID-19 was declared an international public health emergency in January, and a pandemic in March of 2020. There are over 23 million confirmed COVID-19 cases that have cause over 800 thousand deaths worldwide as of August 19th, 2020. COVID-19 is caused by the SARS-CoV-2 virus. SARS-CoV-2 presents a surface "spike" protein that binds to the ACE2 receptor to infect host cells. In addition to the respiratory tract, SARS-Cov-2 can also infect cells of the oral mucosa, which also express the ACE2 receptor. The spike and ACE2 proteins are highly glycosylated with sialic acid modifications that direct viral-host interactions and infection. Maackia amurensis seed lectin (MASL) has a strong affinity for sialic acid modified proteins and can be used as an antiviral agent. Here, we report that MASL targets the ACE2 receptor, decreases ACE2 expression and glycosylation, suppresses binding of the SARS-CoV-2 spike protein, and decreases expression of inflammatory mediators by oral epithelial cells that cause ARDS in COVID-19 patients. This work identifies MASL as an agent with potential to inhibit SARS-CoV-2 infection and COVID-19 related inflammatory syndromes.
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http://dx.doi.org/10.21203/rs.3.rs-93851/v1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7587785PMC
October 2020

human stem cell derived cultures to monitor calcium signaling in neuronal development and function.

Wellcome Open Res 2020 3;5:16. Epub 2020 Feb 3.

Cellular Organization and Signalling, National Centre for Biological Sciences - TIFR, Bangalore, Karnataka, 560065, India.

The development of the human brain involves multiple cellular processes including cell division, migration, and dendritic growth. These processes are triggered by developmental cues and lead to interactions of neurons and glial cells to derive the final complex organization of the brain. Developmental cues are transduced into cellular processes through the action of multiple intracellular second messengers including calcium. Calcium signals in cells are shaped by large number of proteins and mutations in several of these have been reported in human patients with brain disorders.  However, the manner in which such mutations impact human brain development remains poorly understood. A key limitation in this regard is the need for a model system in which calcium signaling can be studied in neurons of patients with specific brain disorders. Here we describe a protocol to differentiate human neural stem cells into cortical neuronal networks that can be maintained as live cultures up to 120 days in a dish. Our protocol generates a 2D culture that exhibits molecular features of several layers of the human cerebral cortex. Using fluorescence imaging of intracellular calcium levels, we describe the development of neuronal activity as measured by intracellular calcium transients during development . These transients were dependent on the activity of voltage gated calcium channels and were abolished by blocking sodium channel activity. Using transcriptome analysis, we describe the full molecular composition of such cultures following differentiation thus offering an insight into the molecular basis of activity. Our approach will facilitate the understanding of calcium signaling defects during cortical neuron development in patients with specific brain disorders and a mechanistic analysis of these defects using genetic manipulations coupled with cell biological and physiological analysis.
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http://dx.doi.org/10.12688/wellcomeopenres.15626.1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7076282PMC
February 2020

Phosphoinositides: Regulators of Nervous System Function in Health and Disease.

Front Mol Neurosci 2019 23;12:208. Epub 2019 Aug 23.

National Centre for Biological Sciences-TIFR, Bengaluru, India.

Phosphoinositides, the seven phosphorylated derivatives of phosphatidylinositol have emerged as regulators of key sub-cellular processes such as membrane transport, cytoskeletal function and plasma membrane signaling in eukaryotic cells. All of these processes are also present in the cells that constitute the nervous system of animals and in this setting too, these are likely to tune key aspects of cell biology in relation to the unique structure and function of neurons. Phosphoinositides metabolism and function are mediated by enzymes and proteins that are conserved in evolution, and analysis of knockouts of these in animal models implicate this signaling system in neural function. Most recently, with the advent of human genome analysis, mutations in genes encoding components of the phosphoinositide signaling pathway have been implicated in human diseases although the cell biological basis of disease phenotypes in many cases remains unclear. In this review we evaluate existing evidence for the involvement of phosphoinositide signaling in human nervous system diseases and discuss ways of enhancing our understanding of the role of this pathway in the human nervous system's function in health and disease.
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http://dx.doi.org/10.3389/fnmol.2019.00208DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6716428PMC
August 2019

Functional analysis of the biochemical activity of mammalian phosphatidylinositol 5 phosphate 4-kinase enzymes.

Biosci Rep 2019 02 19;39(2). Epub 2019 Feb 19.

National Centre for Biological Sciences, TIFR-GKVK Campus, Bellary Road, Bangalore 560065, India

Phosphatidylinositol 5 phosphate 4-kinase (PIP4K) are enzymes that catalyse the phosphorylation of phosphatidylinositol 5-phosphate (PI5P) to generate PI(4,5)P Mammalian genomes contain three genes, and and murine knockouts for these suggested important physiological roles The proteins encoded by and show widely varying specific activities ; PIP4K2A is highly active and PIP4K2C 2000-times less active, and the relationship between this biochemical activity and function is unknown. By contrast, the genome encodes a single PIP4K (dPIP4K) that shows high specific activity and loss of this enzyme results in reduced salivary gland cell size We find that the kinase activity of dPIP4K is essential for normal salivary gland cell size Despite their highly divergent specific activity, we find that all three mammalian PIP4K isoforms are able to enhance salivary gland cell size in the PIP4K null mutant implying a lack of correlation between activity measurements and function. Further, the kinase activity of PIP4K2C, reported to be almost inactive , is required for function. Our findings suggest the existence of unidentified factors that regulate PIP4K enzyme activity .
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http://dx.doi.org/10.1042/BSR20182210DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6379509PMC
February 2019

Functional analysis of mammalian phospholipase D enzymes.

Biosci Rep 2018 12 7;38(6). Epub 2018 Dec 7.

National Centre for Biological Sciences-TIFR, GKVK Campus, Bellary Road, Bangalore 560065, India

Phosphatidylcholine (PC)-specific phospholipase D (PLD) hydrolyzes the phosphodiester bond of the PC to generate phosphatidic acid (PA) and regulates several subcellular functions. Mammalian genomes contain two genes encoding distinct isoforms of PLD in contrast with invertebrate genomes that include a single PLD gene. However, the significance of two genes within a genome encoding the same biochemical activity remains unclear. Recently, loss of function in the only PLD gene in was reported to result in reduced PA levels and a PA-dependent collapse of the photoreceptor plasma membrane due to defects in vesicular transport. Phylogenetic analysis reveals that human PLD1 (hPLD1) is evolutionarily closer to dPLD than human PLD2 (hPLD2). In the present study, we expressed hPLD1 and hPLD2 in and found that while reconstitution of hPLD1 is able to completely rescue retinal degeneration in a loss of function dPLD mutant, hPLD2 was less effective in its ability to mediate a rescue. Using a newly developed analytical method, we determined the acyl chain composition of PA species produced by each enzyme. While dPLD was able to restore the levels of most PA species in cells, hPLD1 and hPLD2 each were unable to restore the levels of a subset of unique species of PA. Finally, we found that in contrast with hPLD2, dPLD and hPLD1 are uniquely distributed to the subplasma membrane region in photoreceptors. In summary, hPLD1 likely represents the ancestral PLD in mammalian genomes while hPLD2 represents neofunctionalization to generate PA at distinct subcellular membranes.
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http://dx.doi.org/10.1042/BSR20181690DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6435507PMC
December 2018

Src and podoplanin forge a path to destruction.

Drug Discov Today 2019 01 2;24(1):241-249. Epub 2018 Aug 2.

Department of Molecular Biology and Graduate School of Biomedical Sciences, Rowan University School of Osteopathic Medicine, 2 Medical Center Drive, Stratford, NJ, USA. Electronic address:

Cancer and arthritis present an enormous challenge to society. They share pathogenic pathways that involve extracellular matrix degradation, tissue invasion, and inflammation. Most cancer and arthritis treatments affect normal cell function to cause significant adverse effects in patients. Specific pathways that promote cancer and arthritis progression must be elucidated to design more targeted and effective therapeutics. The Src kinase and podoplanin (PDPN) receptor are upregulated in cancer cells, fibroblasts, synoviocytes, and immune cells that increase tissue invasion and inflammation to promote both cancer and arthritis. In this review, we discuss how Src and PDPN forge a path to tissue destruction, and how they can serve as targets for therapeutics to combat cancer and arthritis.
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http://dx.doi.org/10.1016/j.drudis.2018.07.009DOI Listing
January 2019

Estrogen Receptor α- and β-Interacting Proteins Contain Consensus Secondary Structures: An Insilico Study.

Ann Neurosci 2018 Apr 23;25(1):1-10. Epub 2017 Oct 23.

Department of Zoology, Biochemistry and Molecular Biology Laboratory, Banaras Hindu University, Varanasi, India.

Background: Estrogen receptor (ER)α and ERβ are ligand-activated transcription factors that regulate gene expression by binding to estrogen-responsive elements and interacting with several coregulators through protein-protein interactions. Usually, these coregulators bind to the various conserved and functional domains of the receptor through a consensus LXXLL sequence, although variations can be found. The interaction of receptor domains and the consensus motif can be a possible target for nuclear receptor (NR) pharmacology, since modifications in these are responsible for possible pathogenesis of various diseases.

Purpose: The present study focuses on the secondary structure and conserved domains of the ERα and ERβ interacting proteins, using bioinformatics tools and their relation to the function of the coregulators.

Methods: Bioinformatics-based prediction tools like STRING, PSIPRED, PROTPARAM and Conserved Domain Database (CDD) were used. The prediction tools utilized in this study basically determines the characteristics of a possible coregulator by using an already existing protein as a template and determines the presence of any conserved consensus sequence. Coregulators have been enlisted with the help of NCBI, STRING and iHOP. The secondary structures were analyzed using PSIPRED and conserved domains were determined using CDD.

Results: The analysis of the structure has shown the presence of conserved domains and homology between the various coregulators. Each interacting protein contains conserved domains like the nuclear coactivators' domain, the helix-loop-helix domain and the SRC domain.

Conclusion: Such studies give the characteristic features of ERα and ERβ interacting proteins and maybe useful to determine their family and uses in NR pharmacology in health and diseases.
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http://dx.doi.org/10.1159/000481809DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5981639PMC
April 2018

Podoplanin: An emerging cancer biomarker and therapeutic target.

Cancer Sci 2018 May;109(5):1292-1299

Graduate School of Biomedical Sciences and Department of Molecular Biology, Rowan University School of Osteopathic Medicine, Stratford, NJ, USA.

Podoplanin (PDPN) is a transmembrane receptor glycoprotein that is upregulated on transformed cells, cancer associated fibroblasts and inflammatory macrophages that contribute to cancer progression. In particular, PDPN increases tumor cell clonal capacity, epithelial mesenchymal transition, migration, invasion, metastasis and inflammation. Antibodies, CAR-T cells, biologics and synthetic compounds that target PDPN can inhibit cancer progression and septic inflammation in preclinical models. This review describes recent advances in how PDPN may be used as a biomarker and therapeutic target for many types of cancer, including glioma, squamous cell carcinoma, mesothelioma and melanoma.
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http://dx.doi.org/10.1111/cas.13580DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5980289PMC
May 2018

RDGBα localization and function at membrane contact sites is regulated by FFAT-VAP interactions.

J Cell Sci 2018 01 8;131(1). Epub 2018 Jan 8.

Cellular Organization and Signalling, National Centre for Biological Sciences, TIFR-GKVK Campus, Bellary Road, Bangalore 560065, India

Phosphatidylinositol transfer proteins (PITPs) are essential regulators of PLC signalling. The PI transfer domain (PITPd) of multi-domain PITPs is reported to be sufficient for function, questioning the relevance of other domains in the protein. In photoreceptors, loss of RDGBα, a multi-domain PITP localized to membrane contact sites (MCSs), results in multiple defects during PLC signalling. Here, we report that the PITPd of RDGBα does not localize to MCSs and fails to support function during strong PLC stimulation. We show that the MCS localization of RDGBα depends on the interaction of its FFAT motif with dVAP-A. Disruption of the FFAT motif (RDGB) or downregulation of dVAP-A, both result in mis-localization of RDGBα and are associated with loss of function. Importantly, the ability of the PITPd in full-length RDGB to rescue mutant phenotypes was significantly worse than that of the PITPd alone, indicating that an intact FFAT motif is necessary for PITPd activity Thus, the interaction between the FFAT motif and dVAP-A confers not only localization but also intramolecular regulation on lipid transfer by the PITPd of RDGBα. This article has an associated First Person interview with the first author of the paper.
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http://dx.doi.org/10.1242/jcs.207985DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5818063PMC
January 2018

Role of proteasome-dependent protein degradation in long-term operant memory in Aplysia.

Learn Mem 2017 01 15;24(1):59-64. Epub 2016 Dec 15.

Department of Biological Science, Program in Neuroscience, Florida State University, Tallahassee, Florida 32306-4295, USA.

We investigated the in vivo role of protein degradation during intermediate (ITM) and long-term memory (LTM) in Aplysia using an operant learning paradigm. The proteasome inhibitor MG-132 inhibited the induction and molecular consolidation of LTM with no effect on ITM. Remarkably, maintenance of steady-state protein levels through inhibition of protein synthesis using either anisomycin or rapamycin in conjunction with proteasome inhibition permitted the formation of robust 24 h LTM. Our studies suggest a primary role for proteasomal activity in facilitation of gene transcription for LTM and raise the possibility that synaptic mechanisms are sufficient to sustain 24 h memory.
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http://dx.doi.org/10.1101/lm.043794.116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5159658PMC
January 2017

Differential role of calpain-dependent protein cleavage in intermediate and long-term operant memory in Aplysia.

Neurobiol Learn Mem 2017 Jan 29;137:134-141. Epub 2016 Nov 29.

Department of Biological Science, Program in Neuroscience, Florida State University, Tallahassee, FL 32306-4295, United States.

In addition to protein synthesis, protein degradation or protein cleavage may be necessary for intermediate (ITM) and long-term memory (LTM) to remove molecular constraints, facilitate persistent kinase activity and modulate synaptic plasticity. Calpains, a family of conserved calcium dependent cysteine proteases, modulate synaptic function through protein cleavage. We used the marine mollusk Aplysia californica to investigate the in vivo role of calpains during intermediate and long-term operant memory formation using the learning that food is inedible (LFI) paradigm. A single LFI training session, in which the animal associates a specific netted seaweed with the failure to swallow, generates short (30min), intermediate (4-6h) and long-term (24h) memory. Using the calpain inhibitors calpeptin and MDL-28170, we found that ITM requires calpain activity for induction and consolidation similar to the previously reported requirements for persistent protein kinase C activity in intermediate-term LFI memory. The induction of LTM also required calpain activity. In contrast to ITM, calpain activity was not necessary for the molecular consolidation of LTM. Surprisingly, six hours after LFI training we found that calpain activity was necessary for LTM, although this is a time at which neither persistent PKC activity nor protein synthesis is required for the maintenance of long-term LFI memory. These results demonstrate that calpains function in multiple roles in vivo during associative memory formation.
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http://dx.doi.org/10.1016/j.nlm.2016.11.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6179366PMC
January 2017

Acute Sleep Deprivation Blocks Short- and Long-Term Operant Memory in .

Sleep 2016 Dec 1;39(12):2161-2171. Epub 2016 Dec 1.

Department of Biological Science, Florida State University, Tallahassee, FL.

Study Objectives: Insufficient sleep in individuals appears increasingly common due to the demands of modern work schedules and technology use. Consequently, there is a growing need to understand the interactions between sleep deprivation and memory. The current study determined the effects of acute sleep deprivation on short and long-term associative memory using the marine mollusk , a relatively simple model system well known for studies of learning and memory.

Methods: were sleep deprived for 9 hours using context changes and tactile stimulation either prior to or after training for the operant learning paradigm, learning that food is inedible (LFI). The effects of sleep deprivation on short-term (STM) and long-term memory (LTM) were assessed.

Results: Acute sleep deprivation prior to LFI training impaired the induction of STM and LTM with persistent effects lasting at least 24 h. Sleep deprivation immediately after training blocked the consolidation of LTM. However, sleep deprivation following the period of molecular consolidation did not affect memory recall. Memory impairments were independent of handling-induced stress, as daytime handled control animals demonstrated no memory deficits. Additional training immediately after sleep deprivation failed to rescue the induction of memory, but additional training alleviated the persistent impairment in memory induction when training occurred 24 h following sleep deprivation.

Conclusions: Acute sleep deprivation inhibited the induction and consolidation, but not the recall of memory. These behavioral studies establish as an effective model system for studying the interactions between sleep and memory formation.
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http://dx.doi.org/10.5665/sleep.6320DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5103805PMC
December 2016

Chronic sleep deprivation differentially affects short and long-term operant memory in Aplysia.

Neurobiol Learn Mem 2016 10 20;134 Pt B:349-59. Epub 2016 Aug 20.

Department of Biological Science, Program in Neuroscience, Florida State University, Tallahassee, FL 32306, United States. Electronic address:

The induction, formation and maintenance of memory represent dynamic processes modulated by multiple factors including the circadian clock and sleep. Chronic sleep restriction has become common in modern society due to occupational and social demands. Given the impact of cognitive impairments associated with sleep deprivation, there is a vital need for a simple animal model in which to study the interactions between chronic sleep deprivation and memory. We used the marine mollusk Aplysia californica, with its simple nervous system, nocturnal sleep pattern and well-characterized learning paradigms, to assess the effects of two chronic sleep restriction paradigms on short-term (STM) and long-term (LTM) associative memory. The effects of sleep deprivation on memory were evaluated using the operant learning paradigm, learning that food is inedible, in which the animal associates a specific netted seaweed with failed swallowing attempts. We found that two nights of 6h sleep deprivation occurring during the first or last half of the night inhibited both STM and LTM. Moreover, the impairment in STM persisted for more than 24h. A milder, prolonged sleep deprivation paradigm consisting of 3 consecutive nights of 4h sleep deprivation also blocked STM, but had no effect on LTM. These experiments highlight differences in the sensitivity of STM and LTM to chronic sleep deprivation. Moreover, these results establish Aplysia as a valid model for studying the interactions between chronic sleep deprivation and associative memory paving the way for future studies delineating the mechanisms through which sleep restriction affects memory formation.
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http://dx.doi.org/10.1016/j.nlm.2016.08.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5136466PMC
October 2016

Synchrony and desynchrony in circadian clocks: impacts on learning and memory.

Learn Mem 2015 Sep 18;22(9):426-37. Epub 2015 Aug 18.

Department of Biological Science, Program in Neuroscience, Florida State University, Tallahassee, Florida 32306, USA

Circadian clocks evolved under conditions of environmental variation, primarily alternating light dark cycles, to enable organisms to anticipate daily environmental events and coordinate metabolic, physiological, and behavioral activities. However, modern lifestyle and advances in technology have increased the percentage of individuals working in phases misaligned with natural circadian activity rhythms. Endogenous circadian oscillators modulate alertness, the acquisition of learning, memory formation, and the recall of memory with examples of circadian modulation of memory observed across phyla from invertebrates to humans. Cognitive performance and memory are significantly diminished when occurring out of phase with natural circadian rhythms. Disruptions in circadian regulation can lead to impairment in the formation of memories and manifestation of other cognitive deficits. This review explores the types of interactions through which the circadian clock modulates cognition, highlights recent progress in identifying mechanistic interactions between the circadian system and the processes involved in memory formation, and outlines methods used to remediate circadian perturbations and reinforce circadian adaptation.
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http://dx.doi.org/10.1101/lm.038877.115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4561405PMC
September 2015

Cardiac gene therapy: Recent advances and future directions.

J Control Release 2015 Oct 5;215:101-11. Epub 2015 Aug 5.

Department of Pharmaceutical Sciences, School of Pharmacy, University of Pittsburgh, Pittsburgh, PA, 15261, USA; Department of Bioengineering, Swanson School of Engineering, University of Pittsburgh, Pittsburgh, PA, 15261, USA; McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA, 15219, USA. Electronic address:

Gene therapy has the potential to serve as an adaptable platform technology for treating various diseases. Cardiovascular disease is a major cause of mortality in the developed world and genetic modification is steadily becoming a more plausible method to repair and regenerate heart tissue. Recently, new gene targets to treat cardiovascular disease have been identified and developed into therapies that have shown promise in animal models. Some of these therapies have advanced to clinical testing. Despite these recent successes, several barriers must be overcome for gene therapy to become a widely used treatment of cardiovascular diseases. In this review, we evaluate specific genetic targets that can be exploited to treat cardiovascular diseases, list the important delivery barriers for the gene carriers, assess the most promising methods of delivering the genetic information, and discuss the current status of clinical trials involving gene therapies targeted to the heart.
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http://dx.doi.org/10.1016/j.jconrel.2015.08.001DOI Listing
October 2015

Assessing the carcinogenic potential of low-dose exposures to chemical mixtures in the environment: the challenge ahead.

Authors:
William H Goodson Leroy Lowe David O Carpenter Michael Gilbertson Abdul Manaf Ali Adela Lopez de Cerain Salsamendi Ahmed Lasfar Amancio Carnero Amaya Azqueta Amedeo Amedei Amelia K Charles Andrew R Collins Andrew Ward Anna C Salzberg Annamaria Colacci Ann-Karin Olsen Arthur Berg Barry J Barclay Binhua P Zhou Carmen Blanco-Aparicio Carolyn J Baglole Chenfang Dong Chiara Mondello Chia-Wen Hsu Christian C Naus Clement Yedjou Colleen S Curran Dale W Laird Daniel C Koch Danielle J Carlin Dean W Felsher Debasish Roy Dustin G Brown Edward Ratovitski Elizabeth P Ryan Emanuela Corsini Emilio Rojas Eun-Yi Moon Ezio Laconi Fabio Marongiu Fahd Al-Mulla Ferdinando Chiaradonna Firouz Darroudi Francis L Martin Frederik J Van Schooten Gary S Goldberg Gerard Wagemaker Gladys N Nangami Gloria M Calaf Graeme Williams Gregory T Wolf Gudrun Koppen Gunnar Brunborg H Kim Lyerly Harini Krishnan Hasiah Ab Hamid Hemad Yasaei Hideko Sone Hiroshi Kondoh Hosni K Salem Hsue-Yin Hsu Hyun Ho Park Igor Koturbash Isabelle R Miousse A Ivana Scovassi James E Klaunig Jan Vondráček Jayadev Raju Jesse Roman John Pierce Wise Jonathan R Whitfield Jordan Woodrick Joseph A Christopher Josiah Ochieng Juan Fernando Martinez-Leal Judith Weisz Julia Kravchenko Jun Sun Kalan R Prudhomme Kannan Badri Narayanan Karine A Cohen-Solal Kim Moorwood Laetitia Gonzalez Laura Soucek Le Jian Leandro S D'Abronzo Liang-Tzung Lin Lin Li Linda Gulliver Lisa J McCawley Lorenzo Memeo Louis Vermeulen Luc Leyns Luoping Zhang Mahara Valverde Mahin Khatami Maria Fiammetta Romano Marion Chapellier Marc A Williams Mark Wade Masoud H Manjili Matilde E Lleonart Menghang Xia Michael J Gonzalez Michalis V Karamouzis Micheline Kirsch-Volders Monica Vaccari Nancy B Kuemmerle Neetu Singh Nichola Cruickshanks Nicole Kleinstreuer Nik van Larebeke Nuzhat Ahmed Olugbemiga Ogunkua P K Krishnakumar Pankaj Vadgama Paola A Marignani Paramita M Ghosh Patricia Ostrosky-Wegman Patricia A Thompson Paul Dent Petr Heneberg Philippa Darbre Po Sing Leung Pratima Nangia-Makker Qiang Shawn Cheng R Brooks Robey Rabeah Al-Temaimi Rabindra Roy Rafaela Andrade-Vieira Ranjeet K Sinha Rekha Mehta Renza Vento Riccardo Di Fiore Richard Ponce-Cusi Rita Dornetshuber-Fleiss Rita Nahta Robert C Castellino Roberta Palorini Roslida Abd Hamid Sabine A S Langie Sakina E Eltom Samira A Brooks Sandra Ryeom Sandra S Wise Sarah N Bay Shelley A Harris Silvana Papagerakis Simona Romano Sofia Pavanello Staffan Eriksson Stefano Forte Stephanie C Casey Sudjit Luanpitpong Tae-Jin Lee Takemi Otsuki Tao Chen Thierry Massfelder Thomas Sanderson Tiziana Guarnieri Tove Hultman Valérian Dormoy Valerie Odero-Marah Venkata Sabbisetti Veronique Maguer-Satta W Kimryn Rathmell Wilhelm Engström William K Decker William H Bisson Yon Rojanasakul Yunus Luqmani Zhenbang Chen Zhiwei Hu

Carcinogenesis 2015 Jun;36 Suppl 1:S254-96

Department of Surgery, The Ohio State University College of Medicine, The James Comprehensive Cancer Center, Columbus, OH 43210, USA.

Lifestyle factors are responsible for a considerable portion of cancer incidence worldwide, but credible estimates from the World Health Organization and the International Agency for Research on Cancer (IARC) suggest that the fraction of cancers attributable to toxic environmental exposures is between 7% and 19%. To explore the hypothesis that low-dose exposures to mixtures of chemicals in the environment may be combining to contribute to environmental carcinogenesis, we reviewed 11 hallmark phenotypes of cancer, multiple priority target sites for disruption in each area and prototypical chemical disruptors for all targets, this included dose-response characterizations, evidence of low-dose effects and cross-hallmark effects for all targets and chemicals. In total, 85 examples of chemicals were reviewed for actions on key pathways/mechanisms related to carcinogenesis. Only 15% (13/85) were found to have evidence of a dose-response threshold, whereas 59% (50/85) exerted low-dose effects. No dose-response information was found for the remaining 26% (22/85). Our analysis suggests that the cumulative effects of individual (non-carcinogenic) chemicals acting on different pathways, and a variety of related systems, organs, tissues and cells could plausibly conspire to produce carcinogenic synergies. Additional basic research on carcinogenesis and research focused on low-dose effects of chemical mixtures needs to be rigorously pursued before the merits of this hypothesis can be further advanced. However, the structure of the World Health Organization International Programme on Chemical Safety 'Mode of Action' framework should be revisited as it has inherent weaknesses that are not fully aligned with our current understanding of cancer biology.
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http://dx.doi.org/10.1093/carcin/bgv039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4480130PMC
June 2015

Mechanisms of environmental chemicals that enable the cancer hallmark of evasion of growth suppression.

Carcinogenesis 2015 Jun;36 Suppl 1:S2-18

Environmental and Molecular Toxicology, Environmental Health Sciences Center, Oregon State University, Corvallis, OR 97331, USA.

As part of the Halifax Project, this review brings attention to the potential effects of environmental chemicals on important molecular and cellular regulators of the cancer hallmark of evading growth suppression. Specifically, we review the mechanisms by which cancer cells escape the growth-inhibitory signals of p53, retinoblastoma protein, transforming growth factor-beta, gap junctions and contact inhibition. We discuss the effects of selected environmental chemicals on these mechanisms of growth inhibition and cross-reference the effects of these chemicals in other classical cancer hallmarks.
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http://dx.doi.org/10.1093/carcin/bgv028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4565608PMC
June 2015

PKA and CDK5 can phosphorylate specific serines on the intracellular domain of podoplanin (PDPN) to inhibit cell motility.

Exp Cell Res 2015 Jul 7;335(1):115-22. Epub 2015 May 7.

Graduate School of Biomedical Sciences and Department of Molecular Biology, Rowan University School of Osteopathic Medicine, Science Center, 2 Medical Center Drive, Stratford, NJ 08084, USA. Electronic address:

Podoplanin (PDPN) is a transmembrane glycoprotein that promotes tumor cell migration, invasion, and cancer metastasis. In fact, PDPN expression is induced in many types of cancer. Thus, PDPN has emerged as a functionally relevant cancer biomarker and chemotherapeutic target. PDPN contains 2 intracellular serine residues that are conserved between species ranging from mouse to humans. Recent studies indicate that protein kinase A (PKA) can phosphorylate PDPN in order to inhibit cell migration. However, the number and identification of specific residues phosphorylated by PKA have not been defined. In addition, roles of other kinases that may phosphorylate PDPN to control cell migration have not been investigated. We report here that cyclin dependent kinase 5 (CDK5) can phosphorylate PDPN in addition to PKA. Moreover, results from this study indicate that PKA and CDK5 cooperate to phosphorylate PDPN on both intracellular serine residues to decrease cell motility. These results provide new insight into PDPN phosphorylation dynamics and the role of PDPN in cell motility. Understanding novel mechanisms of PDPN intracellular signaling could assist with designing novel targeted chemotherapeutic agents and procedures.
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http://dx.doi.org/10.1016/j.yexcr.2015.04.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4556139PMC
July 2015

Antibody and lectin target podoplanin to inhibit oral squamous carcinoma cell migration and viability by distinct mechanisms.

Oncotarget 2015 Apr;6(11):9045-60

Departments of Molecular Biology, Cell Biology, and Pathology, School of Osteopathic Medicine, Rowan University, Stratford, NJ, USA.

Podoplanin (PDPN) is a unique transmembrane receptor that promotes tumor cell motility. Indeed, PDPN may serve as a chemotherapeutic target for primary and metastatic cancer cells, particularly oral squamous cell carcinoma (OSCC) cells that cause most oral cancers. Here, we studied how a monoclonal antibody (NZ-1) and lectin (MASL) that target PDPN affect human OSCC cell motility and viability. Both reagents inhibited the migration of PDPN expressing OSCC cells at nanomolar concentrations before inhibiting cell viability at micromolar concentrations. In addition, both reagents induced mitochondrial membrane permeability transition to kill OSCC cells that express PDPN by caspase independent nonapoptotic necrosis. Furthermore, MASL displayed a surprisingly robust ability to target PDPN on OSCC cells within minutes of exposure, and significantly inhibited human OSCC dissemination in zebrafish embryos. Moreover, we report that human OSCC cells formed tumors that expressed PDPN in mice, and induced PDPN expression in infiltrating host murine cancer associated fibroblasts. Taken together, these data suggest that antibodies and lectins may be utilized to combat OSCC and other cancers that express PDPN.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4496201PMC
http://dx.doi.org/10.18632/oncotarget.3515DOI Listing
April 2015

Podoplanin: a marker for reactive gliosis in gliomas and brain injury.

J Neuropathol Exp Neurol 2015 Jan;74(1):64-74

From the Department of Cellular and Physiological Sciences, Life Sciences Institute, The University of British Columbia, Vancouver, British Columbia, Canada (KK, MF-A, JFB, CCN, WCS); and Department of Molecular Biology and Graduate School of Biomedical Sciences, Rowan University School of Medicine, Stratford, New Jersey (HK, GSG).

Reactive astrogliosis is associated with many pathologic processes in the central nervous system, including gliomas. The glycoprotein podoplanin (PDPN) is upregulated in malignant gliomas. Using a syngeneic intracranial glioma mouse model, we show that PDPN is highly expressed in a subset of glial fibrillary acidic protein-positive astrocytes within and adjacent to gliomas. The expression of PDPN in tumor-associated reactive astrocytes was confirmed by its colocalization with the astrocytic marker S100β and with connexin43, a major astrocytic gap junction protein. To determine whether the increase in PDPN is a general feature of gliosis, we used 2 mouse models in which astrogliosis was induced either by a needle injury or ischemia and observed similar upregulation of PDPN in reactive astrocytes in both models. Astrocytic PDPN was also found to be coexpressed with nestin, an intermediate filament marker for neural stem/progenitor cells. Our findings confirm that expression of PDPN is part of the normal host response to brain injury and gliomas, and suggest that it may be a novel cell surface marker for a specific population of reactive astrocytes in the vicinity of gliomas and nonneoplastic brain lesions. The findings also highlight the heterogeneity of glial fibrillary acidic protein-positive astrocytes in reactive gliosis.
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http://dx.doi.org/10.1097/NEN.0000000000000150DOI Listing
January 2015

Characterization of sleep in Aplysia californica.

Sleep 2014 Sep 1;37(9):1453-63. Epub 2014 Sep 1.

Study Objective: To characterize sleep in the marine mollusk, Aplysia californica.

Design: Animal behavior and activity were assessed using video recordings to measure activity, resting posture, resting place preference, and behavior after rest deprivation. Latencies for behavioral responses were measured for appetitive and aversive stimuli for animals in the wake and rest states.

Setting: Circadian research laboratory for Aplysia.

Patients Or Participants: A. californica from the Pacific Ocean.

Interventions: N/A.

Measurements And Results: Aplysia rest almost exclusively during the night in a semi-contracted body position with preferential resting locations in the upper corners of their tank. Resting animals demonstrate longer latencies in head orientation and biting in response to a seaweed stimulus and less frequent escape response steps following an aversive salt stimulus applied to the tail compared to awake animals at the same time point. Aplysia exhibit rebound rest the day following rest deprivation during the night, but not after similar handling stimulation during the day.

Conclusions: Resting behavior in Aplysia fulfills all invertebrate characteristics of sleep including: (1) a specific sleep body posture, (2) preferred resting location, (3) reversible behavioral quiescence, (4) elevated arousal thresholds for sensory stimuli during sleep, and (5) compensatory sleep rebound after sleep deprivation.
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http://dx.doi.org/10.5665/sleep.3992DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4153062PMC
September 2014

DOR - a Database of Olfactory Receptors - Integrated Repository for Sequence and Secondary Structural Information of Olfactory Receptors in Selected Eukaryotic Genomes.

Bioinform Biol Insights 2014 12;8:147-58. Epub 2014 Jun 12.

National Center for Biological Sciences (TIFR), Bangalore, India.

Olfaction is the response to odors and is mediated by a class of membrane-bound proteins called olfactory receptors (ORs). An understanding of these receptors serves as a good model for basic signal transduction mechanisms and also provides important clues for the strategies adopted by organisms for their ultimate survival using chemosensory perception in search of food or defense against predators. Prior research on cross-genome phylogenetic analyses from our group motivated the addressal of conserved evolutionary trends, clustering, and ortholog prediction of ORs. The database of olfactory receptors (DOR) is a repository that provides sequence and structural information on ORs of selected organisms (such as Saccharomyces cerevisiae, Drosophila melanogaster, Caenorhabditis elegans, Mus musculus, and Homo sapiens). Users can download OR sequences, study predicted membrane topology, and obtain cross-genome sequence alignments and phylogeny, including three-dimensional (3D) structural models of 100 selected ORs and their predicted dimer interfaces. The database can be accessed from http://caps.ncbs.res.in/DOR. Such a database should be helpful in designing experiments on point mutations to probe into the possible dimerization modes of ORs and to even understand the evolutionary changes between different receptors.
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http://dx.doi.org/10.4137/BBI.S14858DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4069036PMC
July 2014

Estrogen modulates in vitro T cell responses in a concentration- and receptor-dependent manner: effects on intracellular molecular targets and antioxidant enzymes.

Mol Immunol 2013 Dec 1;56(4):328-39. Epub 2013 Aug 1.

Integrative Medicine Laboratory, Department of Biotechnology, School of Bioengineering, SRM University, Kattankulathur 603203 Tamil Nadu, India.

Estrogen is a key hormone in facilitating ovulation and maintenance of pregnancy in young females and subsequent decline in its production contributes to the development of age-associated disorders such as hormone-dependent cancer, osteoporosis, and cardiovascular diseases. The mechanisms through which estrogen promotes female-specific diseases with advancing age are unclear especially, its effects on immune system which is vital for the maintenance of homeostasis and health. Although the diverse effects of estrogen on Th immunity (Th1 vs. Th2) have been characterized in several cell-types and animal models, there is no direct mechanistic study to understand its immunomodulatory actions. The purpose of this study is to investigate whether the in vitro effects of 17β-estradiol on lymphocytes from the spleen influence cell-mediated immune responses based on its concentration and type of estrogen receptors (ERs) and to assess its mechanism of action at the cellular level. Lymphocytes from the spleens of young Sprague-Dawley rats were isolated and incubated with various concentrations of 17β-estradiol (10(-6)-10(-14)M) and specific ERα- and β-agonists (10(-6)M, 10(-8)M and 10(-10)M) without or with concanavalin A (Con A) to measure T lymphocyte proliferation, IFN-γ and IL-2 production, p-ERK 1/2, p-CREB, and p-Akt, activities of antioxidant enzymes[superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx)], and nitric oxide (NO) production. The specificity of ER-mediated actions in lymphocytes was examined by coincubation with nonspecific ER antagonists ICI(182,780) or tamoxifen. Lower concentrations of 17β-estradiol enhanced proliferation of T lymphocytes and IFN-γ production without or with Con A stimulation but had no effect on IL-2 production. ERα and ERβ agonists induced an increase in T cell proliferation and IFN-γ production and these effects were inhibited by tamoxifen. ERβ agonist alone enhanced IL-2 production by the lymphocytes. Coincubation with 17β-estradiol and ERα- and β-agonists augmented p-ERK 1/2, p-CREB, and p-Akt expression in the lymphocytes and tamoxifen reversed the ER agonist-induced effects on these molecular targets. Estrogen increased the activities of SOD, CAT, and GPx in both non-stimulated and Con A-stimulated splenocytes in a concentration-dependent manner. Both ERα- and β-agonists enhanced CAT and GPx activity while ERα-agonist decreased SOD activity and ERβ-agonist increased SOD activity. The effects of ER agonists on the antioxidant enzymes were reversed by ICI(182,780). Coincubation of lower doses of 17β-estradiol with Con A and both ER agonists enhanced NO production while higher dose of estrogen with Con A and ERα agonist suppressed its production and these effects were reversed by tamoxifen. Taken together, these results suggest that the effects of estrogen on the cell-mediated immune responses are dependent upon its concentrations and mediated through specific estrogen receptors involving intracellular signaling pathways and antioxidant enzymes.
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http://dx.doi.org/10.1016/j.molimm.2013.05.226DOI Listing
December 2013

Serines in the intracellular tail of podoplanin (PDPN) regulate cell motility.

J Biol Chem 2013 Apr 25;288(17):12215-21. Epub 2013 Mar 25.

Graduate School of Biomedical Sciences and Department of Molecular Biology, School of Osteopathic Medicine, University of Medicine and Dentistry of New Jersey, Stratford, New Jersey 08084, USA.

Podoplanin (PDPN) is a transmembrane receptor that affects the activities of Rho, ezrin, and other proteins to promote tumor cell motility, invasion, and metastasis. PDPN is found in many types of cancer and may serve as a tumor biomarker and chemotherapeutic target. The intracellular region of PDPN contains only two serines, and these are conserved in mammals including mice and humans. We generated cells from the embryos of homozygous null Pdpn knock-out mice to investigate the relevance of these serines to cell growth and migration on a clear (PDPN-free) background. We report here that one or both of these serines can be phosphorylated by PKA (protein kinase A). We also report that conversion of these serines to nonphosphorylatable alanine residues enhances cell migration, whereas their conversion to phosphomimetic aspartate residues decreases cell migration. These results indicate that PKA can phosphorylate PDPN to decrease cell migration. In addition, we report that PDPN expression in fibroblasts causes them to facilitate the motility and viability of neighboring melanoma cells in coculture. These findings shed new light on how PDPN promotes cell motility, its role in tumorigenesis, and its utility as a functionally relevant biomarker and chemotherapeutic target.
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http://dx.doi.org/10.1074/jbc.C112.446823DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3636905PMC
April 2013

SRC points the way to biomarkers and chemotherapeutic targets.

Genes Cancer 2012 May;3(5-6):426-35

University of Medicine and Dentistry of New Jersey, Graduate School of Biomedical Sciences, School of Osteopathic Medicine, Stratford, NJ, USA.

The role of Src in tumorigenesis has been extensively studied since the work of Peyton Rous over a hundred years ago. Src is a non-receptor tyrosine kinase that plays key roles in signaling pathways controlling tumor cell growth and migration. Src regulates the activities of numerous molecules to induce cell transformation. However, transformed cells do not always migrate and realize their tumorigenic potential. They can be normalized by surrounding nontransformed cells by a process called contact normalization. Tumor cells need to override contact normalization to become malignant or metastatic. In this review, we discuss the role of Src in cell migration and contact normalization, with emphasis on Cas and Abl pathways. This paradigm illuminates several chemotherapeutic targets and may lead to the identification of new biomarkers and the development of effective anticancer treatments.
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http://dx.doi.org/10.1177/1947601912458583DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3513789PMC
May 2012

Plant lectin can target receptors containing sialic acid, exemplified by podoplanin, to inhibit transformed cell growth and migration.

PLoS One 2012 23;7(7):e41845. Epub 2012 Jul 23.

Graduate School of Biomedical Sciences, University of Medicine and Dentistry of New Jersey, Stratford, New Jersey, United States of America.

Cancer is a leading cause of death of men and women worldwide. Tumor cell motility contributes to metastatic invasion that causes the vast majority of cancer deaths. Extracellular receptors modified by α2,3-sialic acids that promote this motility can serve as ideal chemotherapeutic targets. For example, the extracellular domain of the mucin receptor podoplanin (PDPN) is highly O-glycosylated with α2,3-sialic acid linked to galactose. PDPN is activated by endogenous ligands to induce tumor cell motility and metastasis. Dietary lectins that target proteins containing α2,3-sialic acid inhibit tumor cell growth. However, anti-cancer lectins that have been examined thus far target receptors that have not been identified. We report here that a lectin from the seeds of Maackia amurensis (MASL) with affinity for O-linked carbohydrate chains containing sialic acid targets PDPN to inhibit transformed cell growth and motility at nanomolar concentrations. Interestingly, the biological activity of this lectin survives gastrointestinal proteolysis and enters the cardiovascular system to inhibit melanoma cell growth, migration, and tumorigenesis. These studies demonstrate how lectins may be used to help develop dietary agents that target specific receptors to combat malignant cell growth.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0041845PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3402461PMC
November 2012

Src activates Abl to augment Robo1 expression in order to promote tumor cell migration.

Oncotarget 2010 Jul;1(3):198-209

Molecular Biology Department, University of Medicine and Dentistry of New Jersey, Stratford, NJ 08084, USA.

Cell migration is an essential step in cancer invasion and metastasis. A number of orchestrated cellular events involving tyrosine kinases and signaling receptors enable cancer cells to dislodge from primary tumors and colonize elsewhere in the body. For example, activation of the Src and Abl kinases can mediate events that promote tumor cell migration. Also, activation of the Robo1 receptor can induce tumor cell migration. However, while the importance of Src, Abl, and Robo1 in cell migration have been demonstrated, molecular mechanisms by which they collectively influence cell migration have not been clearly elucidated. In addition, little is known about mechanisms that control Robo1 expression. We report here that Src activates Abl to stabilize Robo1 in order to promote cell migration. Inhibition of Abl kinase activity by siRNA or kinase blockers decreased Robo1 protein levels and suppressed the migration of transformed cells. We also provide evidence that Robo1 utilizes Cdc42 and Rac1 GTPases to induce cell migration. In addition, inhibition of Robo1 signaling can suppress transformed cell migration in the face of robust Src and Abl kinase activity. Therefore, inhibitors of Src, Abl, Robo1 and small GTPases may target a coordinated pathway required for tumor cell migration.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3058788PMC
http://dx.doi.org/10.18632/oncotarget.100710DOI Listing
July 2010