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Expression of Phospholipase C-beta Isoenzyme in Embryonic Mice
No full textPhospholipase C enzymes hydrolyze the rare membrane lipids phosphatidylinositol-4-phosphate (PIP) and phosphatidylinositol-4,5-bisphosphate (PIP2) PIP into inositol triphosphate (IP3) and diacylglycerol (DAG). Phospholipase C enzymes of the β subtype (PLC-β) function specifically in the signal transduction pathways regulated by Gαq-coupled seven transmembrane receptors. These signaling pathways link plasma membrane receptor activation to intracellular increases in Ca++ through liberation of IP3, activation of protein kinases C through hydrolytic production of DAG, and regulation of ion channels by reduction of PIP2 levels. Second messengers in this bifurcating signaling pathway are utilized ubiquitously to activate physiologic processes as divergent as prostaglandin production, neutrophil chemotaxis, and smooth contraction among many other effects (1).
The PLC-β family is composed of gene products from four genetic loci (PLC-β1, -β2, -β3, and β3) with two splice variants from PLC-β1, a and b, that diverge at the C terminus (1). Most tissues express more than one isoform, with PLC-β1 and PLC-β3 being most ubiquitously expressed (2-4); PLC-β4 being most highly expressed in brain (4), and PLC-β2 (2,3) being more restricted to hematopoietic cells. Functional differences between the isoforms include differential sensitivity to activation by Gαq and Gβγ subunits. All known isoforms are activated by Gαq and increased cytosolic Ca++ with varying affinities (2), but only the PLC-β2 and PLC-β3 isoforms appear to be regulated by Gβγ subunits in a physiological context (5).
Our interest is the role of PLC-β in regulation of cardiomyocyte hypertrophy in atrial dilatation leading to atrial fibrillation and in ventricular hypertrophy in heart failure. Mouse models and human disease investigations link activation of PLC-β to atrial fibrillation and cardiomyocyte hypertrophy (6,7). Recent studies also link the prenatal intrauterine environment to susceptibility to heart disease in adult animals (8). We are interested in the linkage between heart development and heart disease. In the process of characterizing alterations in PLC-β isoenzymes in heart disease models, we realized that little to no information existed on expression of PLC-β isoforms in embryonic heart development. Therefore, we sought to compare protein and mRNA expression levels of PLCβ1-4 in mice hearts at different developmental stages, specifically E10.5, E 12.5, neonate and weanling (P21) adult.
The mRNA and protein expression levels of PLC-β isoforms were compared. PLC-β1, PLC-β3, and PLC-β4, both protein and mRNA, were expressed in E10.5, E12.5 and adult mouse hearts. The mRNA expression level of PLC-β3 was higher than the combined expression of PLCβ1a and PLCβ1b at every developmental age. PLC-β4 mRNA levels were approximately the same as the combined levels of PLC-β1a and PLC-β1b.
We were seeing the same expression pattern of all PLCβs isoforms throughout development in mouse hearts. Previous studies on rat neonatal cardiomyocytes in culture or from excised hearts had similar, if not identical findings. Both PLC-β1 and PLC-β3 proteins were found in rat neonatal cardiomyocytes expressed at approximately the same level (10). Study on rats shown that PLC-β1 and PLC-β3 mRNA and proteins were both expressed in hearts. The mRNA and protein levels of PLC-β3 were expressed higher than PLC-β1 (11), which supports our findings. Protein expression of PLC-β1 and PLC-β3 in rodent hearts found that PLC-β3 was expressed but PLCβ1 was not (12). A study on development of rat hearts from day 3-112 showed that mRNA and PLC-β1 proteins were expressed. Slight alterations of PLC-β1 mRNA and protein expression were observed but PLC-β1 both mRNA and proteins were constantly expressed throughout development (13).
In summary, we did not observe large differences in expression patterns of PLC-β isoforms through two stages of embryonic heart development, E10 and E12.5, during which the mouse heart is undergoing major structural rearrangements and development to its final architecture. Nor did we observe significant differences in PLC-β expression patterns between embryonic and adult mouse hearts, or between embryonic and rodent neonatal hearts as reported by others. Thus, future studies of heart development and disease will be informed by our observation that PLC-β isoform expression is relatively unchanged across developmental stages and rodent ages
Humulus lupulus L.: Evaluation of Phytochemical Profile and Activation of Bitter Taste Receptors to Regulate Appetite and Satiety in Intestinal Secretin Tumor Cell Line (STC‐1 Cells)
Full text linkScope: Inflorescences of the female hop plant (Humulus lupulus L.) contain biologically active compounds, most of which have a bitter taste. Given the rising global obesity rates, there is much increasing interest in bitter taste receptors (TAS2Rs). Intestinal TAS2Rs can have beneficial effects on obesity when activated by bitter agonists. This study aims to investigate the mechanism of action of a hydroalcoholic hop extract in promoting hormone secretion that reduces the sense of hunger at the intestinal level through the interaction with TAS2Rs. Methods and results: The results demonstrate that the hop extract is a rich source of bitter compounds (mainly α-, β-acids) that stimulate the secretion of anorexigenic peptides (glucagon-like peptide 1 [GLP-1], cholecystokinin [CCK]) in a calcium-dependent manner while reducing levels of hunger-related hormones like ghrelin. This effect is mediated through interaction with TAS2Rs, particularly Tas2r138 and Tas2r120, and through the activation of downstream signaling cascades. Knockdown of these receptors using siRNA transfection and inhibition of Trpm5, Plcβ-2, and other calcium channels significantly reduces the hop-induced calcium response as well as GLP-1 and CCK secretion. Conclusions: This study provides a potential application of H. lupulus extract for the formulation of food supplements with satiating activity capable of preventing or combating obesity
Whole mount immunohistochemistry and in situ hybridization of larval and adult zebrafish dental tissues
No full textAnalysis of Tooth Innervation in Microfluidic Coculture Devices
Full text linkInnervation plays a key role in the development, homeostasis, and regeneration of organs and tissues. However, the mechanisms underlying these phenomena are not well understood yet. In particular, the role of innervation in tooth development and regeneration is neglected. Cocultures constitute a valuable method to investigate and manipulate the interactions between nerve fibers and teeth in a controlled and isolated environment. Microfluidic systems for allow cocultures of neurons and different cell types in their appropriate culture media, while permitting the passage of axons from one compartment to the other. Here we describe how to isolate and coculture developing trigeminal ganglia and tooth germs in a microfluidic coculture system. This protocol describes a simple and flexible way to coculture ganglia/nerves and their target tissues and to study the roles of specific molecules on such interactions in a controlled and isolated environment
How to Build Transcriptional Network Models of Mammalian Pattern Formation
No full textGenetic regulatory networks of sequence specific transcription factors underlie pattern formation in multicellular organisms. Deciphering and representing the mammalian networks is a central problem in development, neurobiology, and regenerative medicine. Transcriptional networks specify intermingled embryonic cell populations during pattern formation in the vertebrate neural tube. Each embryonic population gives rise to a distinct type of adult neuron. The homeodomain transcription factor Lbx1 is expressed in five such populations and loss of Lbx1 leads to distinct respecifications in each of the five populations. allele, respectively. Microarrays were used to show that expression levels of 8% of all transcription factor genes were altered in the respecified pool. These transcription factor genes constitute 20–30% of the active nodes of the transcriptional network that governs neural tube patterning. Half of the 141 regulated nodes were located in the top 150 clusters of ultraconserved non-coding regions. Generally, Lbx1 repressed genes that have expression patterns outside of the Lbx1-expressing domain and activated genes that have expression patterns inside the Lbx1-expressing domain.nalysis, and think that it will be generally useful in discovering and assigning network interactions to specific populations. We discuss how ANCEA, coupled with population partitioning analysis, can greatly facilitate the systematic dissection of transcriptional networks that underlie mammalian patterning
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
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Skin Homeostasis and Wound Repair
No full textIn the animal kingdom, skin exists in diverse forms across different phyla ranging from single layered structure in invertebrates to double layered epidermal-dermal composites in vertebrates. Beside their main function of serving as a primary interface between the body and the environment, they have various specialized functions across different classes, ranging from cutaneous respiration in amphibians and certain kinds of fishes to production of feathers in birds or thermoregulation in mammals, as adaptive to their respective basic survival needs. Despite the vast structural and functional diversity portrayed by skin between classes, the skin architecture within a given animal class is highly conserved, providing scientists with ample scope to study skin and associated ailments employing model organisms. This dissertation is primarily focused on understanding the role of critical skin morphogenesis regulators in mammalian skin homeostasis and associated skin conditions using mouse models and thereby translate those findings in context of humans for future therapeutic use.
COUP-TF Interacting Protein 1 and 2 (BCL11A and BCL11B) are critical C2H2 zinc finger transcription factors well known to be involved in a suite of developmental processes ranging from development of central nervous system, immune system to hematopoiesis and skin morphogenesis. The various chapters of this dissertation explore the role of each of these transcriptional regulators in maintaining skin homeostasis and how their potential perturbation can be associated with various skin conditions ranging from inflammatory skin diseases like eczema and atopic dermatitis (AD) to chronic wound healing outcomes.
Finally, this dissertation brings into light various pivotal cellular targets having the possibility to be manipulated in the near future to achieve desirable healing outcomes. Further, nanofiber-based drug delivery methods have also been explored in this dissertation in relation to our proposed novel treatment regimen to determine their efficacy in humanized models of murine wound healing, in an attempt to enhance clinical transability
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Recombination, resistance, and drug discovery in Chlamydia
No full textThe intracellular life cycle of Chlamydia has been difficult to study due to the inability to genetically manipulate the bacteria. As a result, indirect methods have been employed to study the pathogen and the aspects that contribute to its unique intracellular life cycle. A wide variety of laboratory adapted strains, animal strains, and clinical strains have been used to model different interactions with the host cell and to highlight, in the face of extreme genetic conservation, the molecular subtleties that distinguish each of these strains from one another. The use of sequencing, traditional biochemical analyses of proteins, and expression of chlamydial proteins in heterologous systems have been the stand by approaches for studying the biology in this system. Here we have explored utilities, both inherent to the chlamydial system, and with the aid of high through put technologies, to develop genetic tools in a system without genetics.
Presented here is the early work that lead to the development of a recombination system and establishment of a recombinant strain library that allows for large scale genotype-phenotype analyses in Chlamydia. We have characterized the recombination events between two interspecies crosses with different antibiotic resistant markers and pioneered the use of next gen sequencing to do full genome characterization to identify regions targeted by recombination. Through these studies, we show that large regions of the chlamydial genome recombine at regions specific to the antibiotic selection markers, tetracycline, rifampin, and ofloxacin. These studies also show that large fragments of DNA both insert into recipient genomes, resulting in duplication events, or recombine at homologous regions, resulting in functional replacement of the genetic sequence.
In partnership with SIGA technologies, we conducted a high throughput screen against Chlamydia caviae GPIC to try to identify compounds that inhibited chlamydial growth, blocked host processes important in chlamydial development, and to develop new research tools that can be used in intracellular pathogen research. Five compounds were identified that blocked chlamydial growth and two of these compounds also inhibited the growth of Coxiella or Staphylococcus aureus. To understand the resistance determinants associated with the inhibitory properties of these compounds, a chemical genetics approach was utilized in the chlamydial system to select for resistance and genome sequence to identify the resistance-associated mutations. Chlamydia develop resistance to certain antibiotics through the accumulation of mutations when exposed to increasing concentration of drugs in vitro. In these studies, we show that they also mutate in response to stress induced by unknown chemical inhibitors and that the mutations associated with the resistance phenotype suggests potential resistance determinants of the uncharacterized inhibitors.
We have also shown the utility of the chlamydial system as a model for characterizing host cell processes important to intracellular pathogen replication and used this system to aid in the development of a host-specific, broad-spectrum anti-infective compound. The data from these studies also suggests that lipid droplets are widely exploited by both viral and bacterial organisms to support their intracellular replication.
Collectively, this work highlights the genetic tools that are available to study a system without traditional genetic techniques and the use of the chlamydial intracellular life cycle to dissect host processes universally important to intracellular pathogen development
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Endogenous and antiviral RNA silencing pathways in Arabidopsis
Full text linkRNA silencing pathways are required for a wide variety of processes in most
eucaryotes. In plants, small-interfering RNA (siRNA) arising from transposons and other
repetitive sequences is associated with heterochromatin formation and maintenance.
MicroRNAs and trans-acting siRNAs encoded at discrete loci function as negative regulators of
gene expression by triggering cleavage or translational repression of mRNA transcripts with
base complementarity to the small RNA. siRNA processed from viral RNA directs antiviral
silencing that represses virus accumulation in plants and other organisms. Together, these
pathways serve numerous functions in plants including genome maintenance, developmental
timing and patterning and antiviral defense.
Virus-encoded RNA silencing suppressor proteins are viral pathogenicity factors and
inhibit the antiviral silencing response through interaction with small RNA intermediates. In
this work, small RNA duplex binding was demonstrated for unrelated suppressors from multiple
viruses using molecular biology and biochemistry techniques. Sequestration of virus-derived
siRNA and microRNA/microRNA* duplexes, inhibition of microRNA methylation, and
perturbation of Arabidopsis development was demonstrated for several suppressors using
transgenic approaches and molecular techniques. Suppressor inhibition of antiviral RNA
silencing and endogenous microRNA pathways indicates that small RNA binding is a common
strategy used by unrelated viruses, and suggests that interference with miRNA-directed
processes may be a general feature contributing to pathogenicity of many viruses.
Finally, small RNA preparation and high-throughput sequencing procedures were
developed for profiling small RNA populations in Arabidopsis. Genome-wide profiles of small
RNA from wild-type Arabidopsis thaliana and silencing pathway mutants revealed dynamic
changes in expression of some microRNA families as well as genome-wide distribution patterns
of small RNA in plants. These results establish high-throughput sequencing as a small RNA
profiling tool and provide a comprehensive description of the major small RNA pathways in
Arabidopsis. Together, the results presented here provide a basic understanding of the breadth
of small RNA pathways in plants and show how interference with these pathways by viruses
contributes to virus disease
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