Cellular and Molecular Physiology Commons^™

Diapause And Anhydrobiosis In Embryos Of Artemia Franciscana : Metabolic Depression, Lea Proteins And Water Stress., Steven Handel, Yuvraj Patil, Shumin Li, Nilay Charkraborty, Arpurva Borcar, Michael Menze, Leaf Boswell, Daniel Moore, Mehmet Toner

Faculty Scholarship

Metabolic depression is typically correlated with extended survival of environmental challenge and energy-limitation in early life stages of various invertebrates and vertebrates. Diapause is an ontogenetically-programmed reduction of development and often metabolism seen in many invertebrates. When embryos of Artemia franciscana enter the state of diapause, the overall metabolic depression is estimated to be greater than 99%. These embryos also contain trehalose and express multiple isoforms of Late Embryogenesis Abundant (LEA) proteins, constituents often present in a number of such anhydrobiotic animals. The mRNA levels for LEA proteins are highest in diapause and post-diapause embryos that possess desiccation tolerance, but …

(R)-Β-Lysine Modified Elongation Factor P Functions In Translation Elongation, Tammy J. Bullwinkle, S. Betty Zou, Andrei Rajkovic, Steven J. Hersch, Sara Elgamal, Nathaniel Robinson, David Smil, Yuri Bolshan, William Wiley Navarre, Michael Ibba

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Post-translational modification of bacterial elongation factor P (EF-P) with (R)-β-lysine at a conserved lysine residue activates the protein in vivo and increases puromycin reactivity of the ribosome in vitro. The additional hydroxylation of EF-P at the same lysine residue by the YfcM protein has also recently been described. The roles of modified and unmodified EF-P during different steps in translation, and how this correlates to its physiological role in the cell, have recently been linked to the synthesis of polyproline stretches in proteins. Polysome analysis indicated that EF-P functions in translation elongation, rather than initiation as proposed previously. This was …

Selection Of Trna Charging Quality Control Mechanisms That Increase Mistranslation Of The Genetic Code, Srujana S. Yadavalli, Michael Ibba

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Mistranslation can follow two events during protein synthesis: production of non-cognate amino acid:transfer RNA (tRNA) pairs by aminoacyl-tRNA synthetases (aaRSs) and inaccurate selection of aminoacyl-tRNAs by the ribosome. Many aaRSs actively edit non-cognate amino acids, but editing mechanisms are not evolutionarily conserved, and their physiological significance remains unclear. To address the connection between aaRSs and mistranslation, the evolutionary divergence of tyrosine editing by phenylalanyl-tRNA synthetase (PheRS) was used as a model. Certain PheRSs are naturally error prone, most notably a Mycoplasma example that displayed a low level of specificity consistent with elevated mistranslation of the proteome. Mycoplasma PheRS was found …

Late Embryogenesis Abundant Proteins Protect Human Hepatoma Cells During Acute Desiccation, Shumin Li, Nilay Chakraborty, Apurva Borcar, Michael A. Menze, Mehmet Toner, Steven C. Hand

Michael Menze

Expression of late embryogenesis abundant (LEA) proteins is highly correlated with desiccation tolerance in anhydrobiotic animals, selected land plants, and bacteria. Genes encoding two LEA proteins, one localized to the cytoplasm/nucleus (AfrLEA2) and one targeted to mitochondria (AfrLEA3m), were stably transfected into human HepG2 cells. A trehalose transporterwas used for intracellular loading of this disaccharide. Cellswere rapidly and uniformly desiccated to low water content (<0.12 g H2O/g dry weight) with a recently developed spin-drying technique. Immediately on rehydration, control cells without LEA proteins or trehalose exhibited 0% membrane integrity, comparedwith 98% in cells loaded with trehalose and expressing AfrLEA2 or AfrLEA3m; surprisingly, AfrLEA3m without trehalose conferred 94% protection. Cell proliferation across 7 d showed an 18-fold increase for cells dried with AfrLEA3m and trehalose, compared with 27-fold for nondried controls. LEA proteins dramatically enhance desiccation tolerance in mammalian cells and offer the opportunity for engineering biostability in the dried state.

Late Embryogenesis Abundant Proteins Protect Human Hepatoma Cells During Acute Desiccation, Shumin Li, Nilay Chakraborty, Apurva Borcar, Michael A. Menze, Mehmet Toner, Steven C. Hand

Faculty Research & Creative Activity

Expression of late embryogenesis abundant (LEA) proteins is highly correlated with desiccation tolerance in anhydrobiotic animals, selected land plants, and bacteria. Genes encoding two LEA proteins, one localized to the cytoplasm/nucleus (AfrLEA2) and one targeted to mitochondria (AfrLEA3m), were stably transfected into human HepG2 cells. A trehalose transporterwas used for intracellular loading of this disaccharide. Cellswere rapidly and uniformly desiccated to low water content (<0.12 g H2O/g dry weight) with a recently developed spin-drying technique. Immediately on rehydration, control cells without LEA proteins or trehalose exhibited 0% membrane integrity, comparedwith 98% in cells loaded with trehalose and expressing AfrLEA2 or AfrLEA3m; surprisingly, AfrLEA3m without trehalose conferred 94% protection. Cell proliferation across 7 d showed an 18-fold increase for cells dried with AfrLEA3m and trehalose, compared with 27-fold for nondried controls. LEA proteins dramatically enhance desiccation tolerance in mammalian cells and offer the opportunity for engineering biostability in the dried state.

Late Embryogenesis Abundant Proteins Protect Human Hepatoma Cells During Acute Desiccation., Shumin Li, Nilay Chakraborty, Apurva Borcar, Michael Menze, Mehmet Toner, Steven Hand

Faculty Scholarship

Expression of late embryogenesis abundant (LEA) proteins is highly correlated with desiccation tolerance in anhydrobiotic animals, selected land plants, and bacteria. Genes encoding two LEA proteins, one localized to the cytoplasm/nucleus (AfrLEA2) and one targeted to mitochondria (AfrLEA3m), were stably transfected into human HepG2 cells. A trehalose transporterwas used for intracellular loading of this disaccharide. Cellswere rapidly and uniformly desiccated to low water content (< 0.12 g H2O/g dry weight) with a recently developed spin-drying technique. Immediately on rehydration, control cells without LEA proteins or trehalose exhibited 0% membrane integrity, comparedwith 98% in cells loaded with trehalose and expressing AfrLEA2 or AfrLEA3m; surprisingly, AfrLEA3m without trehalose conferred 94% protection. Cell proliferation across 7 d showed an 18-fold increase for cells dried with AfrLEA3m and trehalose, compared with 27-fold for nondried controls. LEA proteins dramatically enhance desiccation tolerance in mammalian cells and offer the opportunity for engineering biostability in the dried state.

Metabolic Preconditioning Of Mammalian Cells: Mimetic Agents For Hypoxia Lack Fidelity In Promoting Phosphorylation Of Pyruvate Dehydrogenase, Apurva Borcar, Michael A. Menze, Mehmet Toner, Steven C. Hand

Michael Menze

Induction of HIF-1α by oxygen limitation promotes increased phosphorylation and catalytic depression of mitochondrial pyruvate dehydrogenase (PDH) and an enhanced glycolytic poise in cells. Cobalt chloride and desferrioxamine are widely used as mimics for hypoxia because they increase the levels of HIF-1α. We evaluated the ability of these agents to elicit selected physiological responses to hypoxia as a means to metabolically precondition mammalian cells, but without the detrimental effects of hypoxia. We show that, while CoCl2 does increase HIF-1α in a dose-dependent manner, it unexpectedly and strikingly decreases PDH phosphorylation at E1α sites 1, 2, and 3 (Ser293, Ser300, and …

Metabolic Preconditioning Of Mammalian Cells: Mimetic Agents For Hypoxia Lack Fidelity In Promoting Phosphorylation Of Pyruvate Dehydrogenase, Apurva Borcar, Michael A. Menze, Mehmet Toner, Steven C. Hand

Faculty Research & Creative Activity

Induction of HIF-1α by oxygen limitation promotes increased phosphorylation and catalytic depression of mitochondrial pyruvate dehydrogenase (PDH) and an enhanced glycolytic poise in cells. Cobalt chloride and desferrioxamine are widely used as mimics for hypoxia because they increase the levels of HIF-1α. We evaluated the ability of these agents to elicit selected physiological responses to hypoxia as a means to metabolically precondition mammalian cells, but without the detrimental effects of hypoxia. We show that, while CoCl2 does increase HIF-1α in a dose-dependent manner, it unexpectedly and strikingly decreases PDH phosphorylation at E1α sites 1, 2, and 3 (Ser293, Ser300, and …

Metabolic Preconditioning Of Mammalian Cells : Mimetic Agents For Hypoxia Lack Fidelity In Promoting Phosphorylation Of Pyruvate Dehydrogenase., Apurva Borcar, Michael Menze, Mehmet Toner, Steven Hand

Faculty Scholarship

Induction of HIF-1α by oxygen limitation promotes increased phosphorylation and catalytic depression of mitochondrial pyruvate dehydrogenase (PDH) and an enhanced glycolytic poise in cells. Cobalt chloride and desferrioxamine are widely used as mimics for hypoxia because they increase the levels of HIF-1α. We evaluated the ability of these agents to elicit selected physiological responses to hypoxia as a means to metabolically precondition mammalian cells, but without the detrimental effects of hypoxia. We show that, while CoCl2 does increase HIF-1α in a dose-dependent manner, it unexpectedly and strikingly decreases PDH phosphorylation at E1α sites 1, 2, and 3 (Ser293, Ser300, and …

Halloween 2012 Jack O'Lanterns Trick Or Treat, George Mcnamara

George McNamara

Halloween 2012 makes trick or treating more visual and interactive than in past years.

the download is a ZIP file containing three files.

Print out the (unnumbered) image on as large and nice printer paper as possible - I used glossy 44" wide here in Miami (University of Miami, MillerSchool of Medicine, Calder Library, Biomedical Communications dept - I also made another print on "fabric", also 44" wide to take with me to an HHMI Janelia Farm conference on 'turning images into knowledge' that ends on Oct 31 - might stay up for a second conference, "GFP..." that start Nov …

Mcnamara 20120831fri-20120904tue Cosmic Ray Particles By Ccd Imaging, George Mcnamara

George McNamara

McNamara 20120831Fri-20120904Tue Cosmic Ray Particles by CCD imaging.zip contains image files in support of a Microscopy Today article - please see

http://www.microscopy-today.com/

Endogenous Inhibitor Proteins That Connect Ser/Thr Kinases And Phosphatases In Cell Signaling., Masumi Eto, David L Brautigan

Department of Molecular Physiology and Biophysics Faculty Papers

Protein phosphatase activity acts as a primary determinant of the extent and duration of phosphorylation of cellular proteins in response to physiological stimuli. Ser/Thr protein phosphatase-1 (PP1) belongs to the PPP superfamily, and is associated with regulatory subunits that confer substrate specificity, allosteric regulation, and subcellular compartmentalization. In addition, all eukaryotic cells contain multiple heat-stable proteins that originally were thought to inhibit phosphatase catalytic subunits released from the regulatory subunits, as a fail-safe mechanism. However, discovery of C-kinase-activated PP1 inhibitor, Mr of 17 kDa (CPI-17) required fresh thinking about the endogenous inhibitors as specific regulators of particular phosphatase complexes, acting …

Mutations In Dmrt3 Affect Locomotion In Horses And Spinal Circuit Function In Mice, Lisa S. Andersson, Martin Larhammar, Fatima Memic, Hanna Wootz, Doreen Schwochow, Carl-Johan Rubin, Kalicharan Patra, Thorvaldur Arnason, Lisbeth Wellbring, Göran Hjälm, Freyja Imsland, Jessica Lynn Petersen, Molly E. Mccue, James R. Mickelson, Gus Cothran, Nadav Ahituv, Lars Roepstorff, Sofia Mikko, Anna Vallstedt, Gabriella Lindgren, Leif Andersson, Klas Kullander

Department of Animal Science: Faculty Publications

Locomotion in mammals relies on a central pattern-generating circuitry of spinal interneurons established during development that coordinates limb movement. These networks produce left–right alternation of limbs as well as coordinated activation of flexor and extensor muscles. Here we show that a premature stop codon in the DMRT3 gene has a major effect on the pattern of locomotion in horses. The mutation is permissive for the ability to perform alternate gaits and has a favorable effect on harness racing performance. Examination of wild-type and Dmrt3-null mice demonstrates that Dmrt3 is expressed in the dI6 subdivision of spinal cord neurons, takes …

Taking Aim At The Start Of Translation, Medha Raina, Michael Ibba

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

No abstract provided.

The Distribution And Functional Properties Of Pmld-Linked Cx47 Mutations On Cx47/Cx47 Homotypic And Cx47/Cx43 Heterotypic Gap Junctions, Mi Seong Kim

Electronic Thesis and Dissertation Repository

Gap junctions (GJ) allow direct intercellular communication, and consist of connexins (Cx). In the mammalian central nervous system, oligodendrocytes express Cx29, Cx32 and Cx47, whereas astrocytes express Cx26, Cx30 and Cx43. Cx47/Cx47 GJs couple oligodendrocytes, and Cx47/Cx43 channels are the primary heterotypic GJs at oligodendrocyte/astrocyte junctions. Interestingly, mutations in Cx47 have been linked to central hypomyelinating Pelizaeus Merzbacher-like Disease (PMLD). My aim is to determine the cellular distribution and functional properties of PMLD-associated Cx47 mutants (I46M, G149S, G236R, G236S, M286T, T398I). All mutants were detected at the cell-cell interface. These mutants, except G149S and T398I, showed no electrical coupling in …

Post-Transcriptional Regulation Of Telomerase Reverse Transcriptase (Tert) In Human Embryonic Stem Cells Cultured Under Varying Oxygen Microenvironments, Lida Radan

Electronic Thesis and Dissertation Repository

Recent evidence points to extra-telomeric roles of telomerase in regulating stem cell function. In this study, human embryonic stem cells (hESCs) were cultured in 20% or 2% oxygen for up to 5 days and evaluated for telomerase reverse transcriptase (TERT) expression and telomerase activity. Results showed increased cell survival and maintenance of the undifferentiated state with elevated levels of nuclear TERT in 2% oxygen cultured hESCs despite no significant difference in telomerase activity. Pharmacological inhibition of TERT caused spontaneous differentiation while inhibition of the telomerase RNA component (TERC) did not. RT-PCR analysis revealed variations in transcript levels of full-length and …

Human Cerebrovascular Endothelial Cell Responses In Severe Sepsis, Christopher J. Blom

Electronic Thesis and Dissertation Repository

Sepsis-associated encephalopathy (SAE) is an independent predictor of mortality in severe sepsis patients. Mechanisms of SAE are poorly characterized. Pro-inflammatory mediators are up-regulated in blood plasma of severe sepsis patients and may lead to activation/dysfunction of cerebrovascular endothelial cells (CMEC) of the blood-brain barrier (BBB) – potentially contributing to brain dysfunction. The objective of the present study was to determine the effects/mechanisms of both human severe sepsis blood plasma (hSSP; 20% v/v) and a mixture of 8 cytokines/chemokines that mimicked physiological concentrations of cytokines/chemokines in hSSP (cytomix; SSCM) on human-derived CMEC (hCMEC/D3) activation/dysfunction

in vitro. hSSP-stimulation up-regulated hCMEC/D3 pro-adhesive …

Cosmic Ray Particles Images With Orca-Ii Erg, George Mcnamara

George McNamara

Cosmic ray particles image series acquired using a Hamamatsu ORCA-II ERG scientific grade CCD camera, cooled to -60 C. Each image is a consecutive 600 second (10 minute) exposure time with no light to the camera.

While processing the data, I discoverd that the background changed around planes 25 and 227 (see Excel file and jpeg screenshots), so I also processed only planes 025-227 (203 planes total, 2030 minutes, 33.83 hours). the CCD industry "rule of thumb" for a "typical" CCD sensor (i.e. 1/3" CCD) is that one cosmic ray particle strikes a sensor approximately every 30 seconds (assuming not …

Green Tea Extract Protects Against Fibrogenesis Associated With Nonalcoholic Fatty Liver Disease In Diet-Induced Obese Rats, Allyson M. Bower

Master's Theses

No abstract provided.

Kiss1r Induces Human Mammary Epithelial Cell Invasiveness And Promotes Breast Cancer Cell Migration And Invasion Via Iqgap1, Donna Cvetkovic

Electronic Thesis and Dissertation Repository

Kisspeptins (KP), peptide products of the kisspeptin-1 (KISS1) gene are the endogenous ligands for a G protein-coupled receptor (KISS1R). KISS1 acts as a metastasis suppressor in numerous human cancers. However, recent studies have demonstrated that an increase in KISS1 and KISS1R expression in human breast tumors correlates with higher tumor grade and metastatic potential. We have previously shown that KP-10, the most potent KP, stimulates invasion of estrogen receptor (ER)-negative breast cancer cells via transactivation of the epidermal growth factor receptor (EGFR). Here, I report that KP-10 treatment of the ER-negative non-malignant mammary epithelial MCF10A cells, or stable …