Anatomical And Behavioral Investigation Of C1ql3 In The Mouse Suprachiasmatic Nucleus, David C. Martinelli

UCHC Articles - Research

Many biochemical, physiological, and behavioral processes such as glucose metabolism, body temperature, and sleep-wake cycles show regular daily rhythms. These circadian rhythms are adjusted to the environmental light-dark cycle by a central pacemaker located in the suprachiasmatic nucleus (SCN) in order for the processes to occur at appropriate times of day. Here, we investigated the expression and function of a synaptic organizing protein, C1QL3, in the SCN. We found that C1ql3 is robustly expressed in the SCN. C1ql3 knockout mice have a reduced density of excitatory synapses in the SCN. In addition, these mice exhibited less consolidated activity to the …

Psma Redirects Cell Survival Signaling From The Mapk To The Pi3k-Akt Pathways To Promote The Progression Of Prostate Cancer, Leslie Ann Caromile, Kristina Dortche, M. Mamunur Rahman, Christina L. Grant, Christopher Stoddard, Linda H. Shapiro

UCHC Articles - Research

Increased abundance of the prostate-specific membrane antigen (PSMA) on prostate epithelium is a hallmark of advanced metastatic prostate cancer (PCa) and correlates negatively with prognosis. However, direct evidence that PSMA functionally contributes to PCa progression remains elusive. We generated mice bearing PSMA-positive or PSMA-negative PCa by crossing PSMA-deficient mice with transgenic PCa (TRAMP) models, enabling direct assessment of PCa incidence and progression in the presence or absence of PSMA. Compared with PSMA-positive tumors, PSMA-negative tumors were smaller, lower-grade, and more apoptotic with fewer blood vessels, consistent with the recognized proangiogenic function of PSMA. Relative to PSMA-positive tumors, tumors lacking PSMA …

Tnf And Cd28 Signaling Play Unique But Complementary Roles In The Systemic Recruitment Of Innate Immune Cells After Staphylococcus Aureus Enterotoxin A Inhalation, Julia Svedova, Naomi Tsurutani, Wenhai Liu, Kamal M. Khanna, Anthony T. Vella

UCHC Articles - Research

TNF and CD28 Signaling Play Unique but Complementary Roles in the Systemic Recruitment of Innate Immune Cells after Staphylococcus aureus Enterotoxin A Inhalation.

Cyclin-Dependent Kinase Crk9, Required For Spliced Leader Trans Splicing Of Pre-Mrna In Trypanosomes, Functions In A Complex With A New L-Type Cyclin And A Kinetoplastid-Specific Protein., Nitika Badjatia, Sung Hee Park, Daniela L. Ambrósio, Justin K. Kirkham, Arthur Günzl

UCHC Articles - Research

In eukaryotes, cyclin-dependent kinases (CDKs) control the cell cycle and critical steps in gene expression. The lethal parasite Trypanosoma brucei, member of the phylogenetic order Kinetoplastida, possesses eleven CDKs which, due to high sequence divergence, were generically termed CDC2-related kinases (CRKs). While several CRKs have been implied in the cell cycle, CRK9 was the first trypanosome CDK shown to control the unusual mode of gene expression found in kinetoplastids. In these organisms, protein-coding genes are arranged in tandem arrays which are transcribed polycistronically. Individual mRNAs are processed from precursor RNA by spliced leader (SL) trans splicing and polyadenylation. CRK9 …

Dnah6 And Its Interactions With Pcd Genes In Heterotaxy And Primary Ciliary Dyskinesia, Stephen M. King

UCHC Articles - Research

Heterotaxy, a birth defect involving left-right patterning defects, and primary ciliary dyskinesia (PCD), a sinopulmonary disease with dyskinetic/immotile cilia in the airway are seemingly disparate diseases. However, they have an overlapping genetic etiology involving mutations in cilia genes, a reflection of the common requirement for motile cilia in left-right patterning and airway clearance. While PCD is a monogenic recessive disorder, heterotaxy has a more complex, largely non-monogenic etiology. In this study, we show mutations in the novel dynein gene DNAH6 can cause heterotaxy and ciliary dysfunction similar to PCD. We provide the first evidence that trans-heterozygous interactions between DNAH6 and …

Optimizing Production Of Fc-Amidated Peptides By Chinese Hamster Ovary Cells, Kristina Carlson, Richard E. Mains, Betty A. Eipper

UCHC Articles - Research

Background

Amidation of the carboxyl terminal of many peptides is essential for full biological potency, often increasing receptor binding and stability. The single enzyme responsible for this reaction is peptidylglycine α-amidating monooxygenase (PAM: EC 1.14.17.3), a copper- and ascorbate-dependent Type I membrane protein.

Methods

To make large amounts of high molecular weight amidated product, Chinese hamster ovary (CHO) cells were engineered to express exogenous PAM. To vary access of the enzyme to its substrate, exogenous PAM was targeted to the endoplasmic reticulum, trans-Golgi network, endosomes and lysosomes or to the lumen of the secretory pathway.

Results

PAM was equally …

Phosphorylation State-Dependent Interaction Between Akap7Δ/Γ And Phospholamban Increases Phospholamban Phosphorylation., Marc Rigatti, Andrew V. Le, Claire Gerber, Ion I. Moraru, Kimberly L. Dodge-Kafka

UCHC Articles - Research

Changes in heart rate and contractility in response to sympathetic stimulation occur via activation of cAMP dependent protein kinase A (PKA), leading to phosphorylation of numerous substrates that alter Ca²⁺ cycling. Phosphorylation of these substrates is coordinated by A-kinase anchoring proteins (AKAPs), which recruit PKA to specific substrates [1]. Phosphorylation of the PKA substrate phospholamban (PLB) is a critical determinant of Ca²⁺ re-entry into the sarcoplasmic reticulum and is coordinated by AKAP7δ/γ [2,3]. Here, we further these findings by showing that phosphorylation of PLB requires interaction with AKAP7δ/γ and that this interaction …

Cloning And Variation Of Ground State Intestinal Stem Cells, Lane H. Wilson, Francisco A. Sylvester, Jeffrey S. Hyams, Thomas Devers, Wa Xian

UCHC Articles - Research

Stem cells of the gastrointestinal tract, pancreas, liver, and other columnar epithelia collectively resist cloning in their elemental states. Here we demonstrate the cloning and propagation of highly clonogenic, “ground state” stem cells of the human intestine and colon. We show that derived stem cell pedigrees sustain limited copy number and sequence variation despite extensive serial passaging and display exquisitely precise, cell-autonomous commitment to epithelial differentiation consistent with their origins along the intestinal tract. This developmentally patterned and epigenetically maintained commitment of stem cells likely enforces the functional specificity of the adult intestinal tract. Using clonally-derived colonic epithelia, we show …

Association Of The Lipoprotein Receptor Scarb1 Common Missense Variant Rs4238001 With Incident Coronary Heart Disease, Annabelle Rodriguez-Oquendo

UCHC Articles - Research

Background

Previous studies in mice and humans have implicated the lipoprotein receptor SCARB1 in association with atherosclerosis and lipid levels. In the current study, we sought to examine association of SCARB1 missense single nucleotide polymorphism (SNP) rs4238001 with incident coronary heart disease (CHD).

Methods and Results

Genotypes for rs4238001 were imputed for 2,319 White, 1,570 African American, and 1,292 Hispanic-American MESA participants using the 1,000 Genomes reference set. Cox proportional hazards models were used to determine association of rs4238001 with incident CHD, with adjustments for age, sex, study site, principal components of ancestry, body mass index, diabetes status, serum creatinine, …

Proteo-Lipobeads For The Oriented Encapsulation Of Membrane Proteins, Leslie M. Loew

UCHC Articles - Research

As a surrogate of the life cell, proteo-lipobeads are presented, encapsulating functional membrane proteins in a strict orientation into a lipid bilayer. Assays can be performed just as on life cells, for example using fluorescence measurements. As a proof of concept, we have demonstrated proton transport through cytochrome c oxidase.

Vasculogenesis And Angiogenesis In Vegf Receptor-1 Deficient Mice, Vivienne C. Ho, Guo-Hua Fong

UCHC Articles - Research

Vascular endothelial growth factor receptor-1 (VEGFR-1)/Flt-1 is a transmembrane tyrosine kinase receptor for VEGF-A, VEGF-B, and placental growth factor (PlGF). VEGFR-1 is an enigmatic molecule whose precise role in postnatal angiogenesis remains controversial. Although many postnatal and adult studies have been performed by manipulating VEGFR-1 ligands, including competitive binding by truncated VEGFR-1 protein, neutralization by antibodies, or specific ligand overexpression or knockout, much less is known at the level of the receptor per se, especially in vivo. Perplexingly, while VEGFR-1 negatively regulates endothelial cell differentiation during development, it has been implied in promoting angiogenesis under certain conditions in adult tissues, …

Imaging Submillisecond Membrane Potential Changes From Individual Regions Of Single Axons, Dendrites And Spines, Srdjan D. Antic

UCHC Articles - Research

A central question in neuronal network analysis is how the interaction between individual neurons produces behavior and behavioral modifications. This task depends critically on how exactly signals are integrated by individual nerve cells functioning as complex operational units. Regional electrical properties of branching neuronal processes which determine the input-output function of any neuron are extraordinarily complex, dynamic, and, in the general case, impossible to predict in the absence of detailed measurements. To obtain such a measurement one would, ideally, like to be able to monitor, at multiple sites, subthreshold events as they travel from the sites of origin (synaptic contacts …

Genome-Wide Analysis Of Drosophila Circular Rnas Reveals Their Structural And Sequence Properties And Age-Dependent Neural Accumulation, Sarah Olson, Brenton R. Graveley

UCHC Articles - Research

Circularization was recently recognized to broadly expand transcriptome complexity. Here, we exploit massive Drosophila total RNA-sequencing data, >5 billion paired-end reads from >100 libraries covering diverse developmental stages, tissues, and cultured cells, to rigorously annotate >2,500 fruit fly circular RNAs. These mostly derive from back-splicing of protein-coding genes and lack poly(A) tails, and the circularization of hundreds of genes is conserved across multiple Drosophila species. We elucidate structural and sequence properties of Drosophila circular RNAs, which exhibit commonalities and distinctions from mammalian circles. Notably, Drosophila circular RNAs harbor >1,000 well-conserved canonical miRNA seed matches, especially within coding regions, and coding …

Biphasic Effects Of Fgf2 On Odontoblast Differentiation Involve Changes In The Bmp And Wnt Signaling Pathways, Karen Sagomonyants, Mina Mina

UCHC Articles - Research

Odontoblast differentiation during physiological and reparative dentinogenesis is dependent upon multiple signaling molecules, including Fibroblast Growth Factors (FGFs), Bone Morphogenetic Proteins (BMPs) and Wingless/Integrated (Wnt) ligands. Recent studies in our laboratory showed that continuous exposure of primary dental pulp cultures to FGF2 exerted biphasic effects on the expression of markers of dentinogenesis. In the present study we examined the possible involvement of the BMP and Wnt signaling pathways in mediating the effects of FGF2 on dental pulp cells. Our results showed that stimulatory effects of FGF2 on dentinogenesis during the proliferation phase of growth were associated with increased expression of …

Genetic Determinants Of Amidating Enzyme Activity And Its Relationship With Metal Cofactors In Human Serum, Eric D. Gaier, Alison Kleppinger, Martina Ralle, Jonathan Covault, Richard E. Mains, Anne M. Kenny, Betty A. Eipper

UCHC Articles - Research

Abstract

BACKGROUND:

α-amidation is a final, essential step in the biosynthesis of about half of all peptide hormones and neurotransmitters. Peptidylglycine α-amidating monooxygenase (PAM), with enzymatic domains that utilize Cu and Zn, is the only enzyme that catalyzes this reaction. PAM activity is detected in serum, but its significance and utility as a clinical biomarker remain unexplored.

METHODS:

We used well-established enzymatic assays specific for the peptidylglycine-α -hydroxylating monooxygenase (PHM) and peptidyl-α-hydroxyglycine α-amidating lyase (PAL) domains of PAM to quantify amidating activity in the sera of 144 elderly men. Relationships between PHM and PAL activity and serum levels of their …

Simple Signaling Molecules For Inductive Bone Regenerative Engineering, Bret D. Ulery, Stephen J. Nelson, Meng Deng, Kevin W. H. Lo, Yusuf M. Khan, Cato T. Laurencin

UCHC Articles - Research

Abstract

With greater than 500,000 orthopaedic procedures performed in the United States each year requiring a bone graft, the development of novel graft materials is necessary. We report that some porous polymer/ceramic composite scaffolds possess intrinsic osteoinductivity as shown through their capacity to induce in vivo host osteoid mineralization and in vitro stem cell osteogenesis making them attractive synthetic bone graft substitutes. It was discovered that certain low crystallinity ceramics partially dissociate into simple signaling molecules (i.e., calcium and phosphate ions) that induce stem cells to endogenously produce their own osteoinductive proteins. Review of the literature has uncovered a variety …

Human Esc-Derived Mscs Outperform Bone Marrow Mscs In The Treatment Of An Eae Model Of Multiple Sclerosis, Xiaofang Wang, Kumiko Ijichi, Debayon Paul, Adam S. Lazorchak, Joel S. Pachter, Stephen J. Crocker, Ren-He Xu

UCHC Articles - Research

Current therapies for multiple sclerosis (MS) are largely palliative, not curative. Mesenchymal stem cells (MSCs) harbor regenerative and immunosuppressive functions, indicating a potential therapy for MS, yet the variability and low potency of MSCs from adult sources hinder their therapeutic potential. MSCs derived from human embryonic stem cells (hES-MSCs) may be better suited for clinical treatment of MS because of their unlimited and stable supply. Here, we show that hES-MSCs significantly reduce clinical symptoms and prevent neuronal demyelination in a mouse experimental autoimmune encephalitis (EAE) model of MS, and that the EAE disease-modifying effect of hES-MSCs is significantly greater than …

Increased Mitochondrial Biogenesis Preserves Intestinal Stem Cell Homeostasis And Contributes To Longevity In Indy Mutant Flies, Ryan P. Rogers, Blanka Rogina

UCHC Articles - Research

The Drosophila Indy (I'm Not Dead Yet) gene encodes a plasma membrane transporter of Krebs cycle intermediates, with robust expression in tissues associated with metabolism. Reduced INDY alters metabolism and extends longevity in a manner similar to caloric restriction (CR); however, little is known about the tissue specific physiological effects of INDY reduction. Here we focused on the effects of INDY reduction in the Drosophila midgut due to the importance of intestinal tissue homeostasis in healthy aging and longevity. The expression of Indy mRNA in the midgut changes in response to aging and nutrition. Genetic reduction of Indy expression increases …

Resolution Of Central Nervous System Astrocytic And Endothelial Sources Of Ccl2 Gene Expression During Evolving Neuroinflammation, Bandana Shrestha, Shujun Ge, Joel S. Pachter

UCHC Articles - Research

Background

The chemokine CCL2 is a critical mediator of neuroinflammation in diseases such as multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). CCL2 drives mononuclear cell infiltration into the central nervous system (CNS), alters expression and distribution of microvascular endothelial tight junction proteins, and disrupts the blood–brain and blood-spinal cord barriers. Immunohistochemistry has consistently revealed astrocytes to be a source of this chemokine during neuroinflammation, while providing less uniform evidence that CNS endothelial cells may also express CCL2. Moreover, the relative contributions of these cell types to the CNS pool of CCL2 during MS/EAE are unclear and …

Cell-Selective Knockout And 3d Confocal Image Analysis Reveals Separate Roles For Astrocyte- And Endothelial-Derived Ccl2 In Neuroinflammation, Debayon Paul, Shujun Ge, Yen Lemire, Evan R. Jellison, David R. Serwanski, Joel S. Pachter

UCHC Articles - Research

Background

Expression of chemokine CCL2 in the normal central nervous system (CNS) is nearly undetectable, but is significantly upregulated and drives neuroinflammation during experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis which is considered a contributing factor in the human disease. As astrocytes and brain microvascular endothelial cells (BMEC) forming the blood–brain barrier (BBB) are sources of CCL2 in EAE and other neuroinflammatory conditions, it is unclear if one or both CCL2 pools are critical to disease and by what mechanism(s).

Methods

Mice with selective CCL2 gene knockout (KO) in astrocytes (Astro KO) or …

A Systems Biology Approach To Iron Metabolism, Reinhard C. Laubenbacher, Suzy V. Torti

UCHC Articles - Research

Iron is critical to the survival of almost all living organisms. However, inappropriately low or high levels of iron are detrimental and contribute to a wide range of diseases. Recent advances in the study of iron metabolism have revealed multiple intricate pathways that are essential to the maintenance of iron homeostasis. Further, iron regulation involves processes at several scales, ranging from the subcellular to the organismal. This complexity makes a systems biology approach crucial, with its enabling technology of computational models based on a mathematical description of regulatory systems. Systems biology may represent a new strategy for understanding imbalances in …

Expression Of Phosphodiesterase 6 (Pde6) In Human Breast Cancer Cells, Hongli Dong, Kevin P. Claffey, Stefan Brocke, Paul M. Epstein

UCHC Articles - Research

Considerable epidemiological evidence demonstrates a positive association between artificial light at night (LAN) levels and incidence rates of breast cancer, suggesting that exposure to LAN is a risk factor for breast cancer. There is a 30-50% higher risk of breast cancer in the highest LAN exposed countries compared to the lowest LAN countries, and studies showing higher incidence of breast cancer among shift workers exposed to more LAN have led the International Agency for Research on Cancer to classify shift work as a probable human carcinogen. Nevertheless, the means by which light can affect breast cancer is still unknown. In …

One-Day Treatment Of Small Molecule 8-Bromo-Cyclic Amp Analogue Induces Cell-Based Vegf Production For In Vitro Angiogenesis And Osteoblastic Differentiation, Kevin W.-H. Lo, Ho Man Kan, Keith A. Gagnon, Cato T. Laurencin

UCHC Articles - Research

Small molecule based regenerative engineering is emerging as a promising strategy for regenerating bone tissue. Small molecule cAMP analogues have been proposed as novel biofactors for bone repair and regeneration, and while promising, the effect that these small molecules have on angiogenesis, a critical requirement for successful bone regeneration, is still unclear. Our previous research demonstrated that the small molecule cAMP analogue 8-bromoadenosine-3’,5’-cyclic monophosphate (8-Br-cAMP) was able to promote initial osteoblast adhesion on a polymeric scaffold via cAMP signaling cascades. Here, we report that 8-Br-cAMP is capable of inducing in vitro cell-based VEGF production for angiogenesis promotion. We first demonstrated …

Preosteocytes/Osteocytes Have The Potential To Dedifferentiate Becoming A Source Of Osteoblasts, Elena Torreggiani, Brya G. Matthews, Slavica Pejda, Igor Matic, Danka Grcevic, Mark C. Horowitz, Ivo Kalajzic

UCHC Articles - Research

Presently there is no clear evidence for the ability of mature osteogenic lineage cells to dedifferentiate. In order to identify and trace mature osteogenic lineage cells, we have utilized transgenic mouse models in which the dentin matrix protein 1 (Dmp1) promoter drives expression of GFP (active marker) or Cre recombinase (historic label) in preosteocytes/osteocytes. In long bone chip outgrowth cultures, in which cells on the bone surface were enzymatically removed, cells with previous activity of the Dmp1 promoter migrated onto plastic and down-regulated Dmp1-GFP expression. Dmp1Cre-labeled cells from these cultures had the potential to re-differentiate into the osteogenic lineage, while …

Osterix-Cre Labeled Progenitor Cells Contribute To The Formation And Maintenance Of The Bone Marrow Stroma, Yaling Liu, Sara Strecker, Liping Wang, Mark S. Kronenberg, Wen Wang, David W. Rowe, Peter F. Maye

UCHC Articles - Research

We have carried out fate mapping studies using Osterix-EGFPCre and Osterix-CreERt animal models and found Cre reporter expression in many different cell types that make up the bone marrow stroma. Constitutive fate mapping resulted in the labeling of different cellular components located throughout the bone marrow, whereas temporal fate mapping at E14.5 resulted in the labeling of cells within a region of the bone marrow. The identity of cell types marked by constitutive and temporal fate mapping included osteoblasts, adipocytes, vascular smooth muscle, perineural, and stromal cells. Prolonged tracing of embryonic precursors labeled at E14.5dpc revealed the continued …

Utilization Of Transgenic Models In Evaluation Of Osteogenic Differentiation Of Embryonic Stem Cells, Dario Repic, Elena Torreggiani, Tiziana Franceschetti, Brya G. Matthews, Alexander C. Lichtler, Ivo Kalajzic

UCHC Articles - Research

Previous studies reported that embryonic stem cells (ESCs) can be induced to differentiate into cells showing a mature osteoblastic phenotype by culturing them under osteo-inductive conditions. It is probable that osteogenic differentiation requires that ESCs undergo differentiation through an intermediary step involving a mesenchymal lineage precursor. Based on our previous studies indicating that adult mesenchymal progenitor cells express αSMA, we have generated ESCs from transgenic mice in which an αSMA promoter directs the expression of red fluorescent protein (RFP) to mesenchymal progenitor cells. To track the transition of ESC-derived MSCs into mature osteoblasts, we have utilized a bone-specific fragment of …

Ptpn11 Deletion In A Novel Cartilage Cell Causes Metachondromatosis By Activating Hedgehog Signaling, Qian Wu

UCHC Articles - Research

SHP2, encoded by PTPN11, is required for survival, proliferation and differentiation of various cell types^1,2. Germ line activating mutations in PTPN11 cause Noonan Syndrome, while somatic PTPN11 mutations cause childhood myeloproliferative disease and contribute to some solid tumors. Recently, heterozygous inactivating mutations in PTPN11 were found in metachondromatosis, a rare inherited disorder featuring multiple exostoses, endochondromas, joint destruction and bony deformities^3,4. The detailed pathogenesis of this disorder has remained unclear. Here, we used a conditional knockout allele (Ptpn11^fl) and Cre recombinase (Cre) transgenic mice to delete Ptpn11 specifically in …

The Transcriptomics To Proteomics Of Hair Cell Regeneration: Looking For A Hair Cell In A Haystack, Gopinath Rajadinakaran

UCHC Articles - Research

Mature mammals exhibit very limited capacity for regeneration of auditory hair cells, while all non-mammalian vertebrates examined can regenerate them. In an effort to find therapeutic targets for deafness and balance disorders, scientists have examined gene expression patterns in auditory tissues under different developmental and experimental conditions. Microarray technology has allowed the large-scale study of gene expression profiles (transcriptomics) at whole-genome levels, but since mRNA expression does not necessarily correlate with protein expression, other methods, such as microRNA analysis and proteomics, are needed to better understand the process of hair cell regeneration. These technologies and some of the results of …

Subtelomeric Hotspots Of Aberrant 5-Hydroxymethylcytosine-Mediated Epigenetic Modifications During Reprogramming To Pluripotency, Stormy J. Chamberlain, I-Ping Chen

UCHC Articles - Research

Mammalian somatic cells can be directly reprogrammed into induced pluripotent stem cells (iPSCs) by introducing defined sets of transcription factors. Somatic cell reprogramming involves epigenomic reconfiguration, conferring iPSCs with characteristics similar to embryonic stem cells (ESCs). Human ES cells contain 5-hydroxymethylcytosine (5hmC), which is generated through the oxidation of 5-methylcytosine by the TET enzyme family. Here we show that 5hmC levels increase significantly during reprogramming to human iPSCs mainly due to TET1 activation, and this hydroxymethylation change is critical for optimal epigenetic reprogramming, but does not compromise primed pluripotency. Compared with hES cells, we find iPS cells tend to form …

Mmp-3 Mediates Psychosine-Induced Globoid Cell Formation: Implications For Leukodystrophy Pathology, Kumiko Ijichi, Graham D. Brown, Craig S. Moore, Paige N. Winokur, Roberto Pagarigan, Stephen J. Crocker

UCHC Articles - Research

Globoid cell leukodystrophy (GLD) or Krabbe disease, is a fatal demyelinating disease attributed to mutations in the galactocerebrosidase (GALC) gene. Loss of function mutations in GALC result in accumulation of the glycolipid intermediate, galactosylsphingosine (psychosine). Due to the cytotoxicity of psychosine, it has been hypothesized that accumulated psychosine underlie the pathophysiology of GLD. However, the cellular mechanisms of GLD pathophysiology remain unclear. Globoid cells, multinucleated microglia/macrophages in the central nervous system (CNS), are a defining characteristic of GLD. Here we report that exposure of primary glial cultures to psychosine induces the expression and the production of matrix metalloproteinase …