Dabney-Smith, Carole

Lorigan, Gary

Mayo, Daniel J.

Storm, Amanda R.

Elsevier - Publisher Connector 

Monday, February 4, 2013 343aHere we use a non-invasive method to measure the metabolic phenotype of sin-
gle colon cancer cells in vivo. By using NADH as optical biomarker and the
phasor approach to Fluorescence Lifetime microscopy (FLIM) we identify can-
cer metabolism related to different rates of glycolysis, cell growth and prolif-
eration of cells. We perform label-free Phasor FLIM on living and actively-
perfused xenograft tumors. Colon cancer cells are injected subcutaneously
into immune deficient (NSG) mice and tumors are allowed to grow for three
weeks. The tumor vasculature is labeled by injecting TRITC Dextran into
the tail vein and xenografts are exposed via a skin flap, still perfused by their
feeder vessels.
FLIM distinguishes collagen fibers, the tumor stroma, adipocytes, blood vessels
and single cancer cells within the living tumor. By measuring NADH lifetime,
we quantify the relative concentration of free and bound NADH in single can-
cer cells, which reflects the cellular redox NADH/NADþ ratio and balance of
oxidative phosphorylation and glycolysis. We investigate the tumor microenvi-
ronment by characterizing the distribution of the metabolic fingerprint of single
colon cancer cells and by mapping the three-dimensional metabolic heteroge-
neity of the tumor at different distances from blood vessels.
Our method permits a non invasive measurement of single cancer cell metab-
olism in a living intact tumor microenvironment. It allows monitoring of spatial
and temporal dynamic changes of tumor metabolism upon different physiolog-
ical conditions such as blood flow, tissue oxygenation levels, nutrient availabil-
ity and drug delivery.
Work supported by NIH-P41 P41-RRO3155, P50-GM076516, NIH RO1,
HD49488, NIH PO1 HD47675.
1757-Pos Board B649
Quantitative Intra-Vital FRET Imaging Reveals Enhanced Sympathetic
Adrenergic Tone in Femoral Arteries in Chronic Angiotensin Ii-Infused
Biosensor Mice
Youhua Wang1, Jingyu Wang1, Ling Chen2, Jin Zhang1.
1Department of Physiology, University of Maryland at Baltimore, Baltimore,
MD, USA, 2Departments of Medicine and Physiology, University of
Maryland at Baltimore, Baltimore, MD, USA.
Hypertension only exists in living animals. Administration of Angiotensin II
(Ang II) to animals increases blood pressure. One of the major effects of
Ang II is to cause vasoconstriction via directly activating the Ang II type-1 re-
ceptor (AT1R) in blood vessels. The aim of this study is to determine whether or
not chronic Ang II infusion evokes conduit arterial constriction in vivo, and if
so what the underlying mechanisms are. Ang II (400 ng/kg/min) or vehicle (sa-
line) was continuously infused for 2 weeks by subcutaneously implanted os-
motic mini-pumps in exogenous myosin light chain kinase FRET biosensor
mice. Blood pressure, measured with an intra-carotid artery catheter under iso-
flurane anesthesia, was significantly elevated in Ang II-infused mice. Mid-thigh
femoral arteries were slightly dissected and superfused with physiological sa-
line solution. Simultaneous in vivo arterial diameter and quantitative cytoplas-
mic [Ca2þ] in smooth muscle cells within intact arteries were measured. Under
resting condition, femoral arteries from Ang II-infused mice exhibited signifi-
cantly increased basal tone and cytoplasmic [Ca2þ]. Local application, in the
superfusion solution, of phentolamine (1 mM), a non-selective a adrenoceptor
antagonist, and prazosin (100 nM), a selective a1 adrenoceptor antagonist,
caused significantly more relaxation and [Ca2þ] reduction in Ang II than in ve-
hicle group. Dose-dependent vasoconstriction induced by an a adrenoceptor
agonist, phenylephrine, however, was similar, suggesting a lack of expression
or sensitivity change of the local adrenoceptor. In contrast, acute Ang II-
induced vasoconstriction was reduced in chronic Ang II-infused mice, suggest-
ing a possible down-regulation of AT1R. These results indicate that, in chronic
Ang II-infused mice, albeit the reduced acute vasoconstrictor response to Ang
II, there is an increased sympathetic adrenergic tone of femoral arteries, which
correlates with blood pressure elevation. (AHA SDG0735461N, NIH R01
HL107654).
EPR Spectroscopy
1758-Pos Board B650
Dynamics of Histone Tails within Chromatin
Morgan W. Bernier, Jaime Bravo, Casey Branson, Christopher P. Jaroniec,
P.C. Hammel, Michael G. Poirier.
The Ohio State University, Columbus, OH, USA.
The physical and material properties of chromatin appear to influence gene ex-
pression by altering the accessibility of proteins to the DNA. The fundamental
unit of chromatin, the nucleosome, consists of DNAwrapped around a core his-
tone octamer protein. The tails of the histone proteins are flexible domains that
are thought to play a role in regulating DNA accessibility and compaction;
however the molecular mechanisms for these are not understood. using SiteDirected Spin Labeling and Electron Paramagnetic Resonance (CW-EPR),
we probe the dynamics of the histone tails within chromatin. The goal of this
study is to further understand the function of histone tails and the part they
play in gene regulation.
1759-Pos Board B651
Sequence-Dependent Local Environment in P53 Response Element Probed
by Site-Directed Spin Labeling
Yuan Ding, Xiaojun Zhang, Peter Qin.
University of Southern California, Los Angeles, CA, USA.
The p53 response elements (RE) are a family of DNAs that are specifically rec-
ognized by the tumor suppressor protein p53. Binding of p53 to REs leads to
transcription activation or suppression of a diverse set of genes that regulate
a number of key biological processes. The molecular basis of p53-RE interac-
tion has been a topic of intensive studies, however, it remains unclear how this
one p53 protein can specifically recognize a large number of DNA targets in
order to elicit diverse functions. As sequence dependent properties of DNAs
are a key determinant in protein-DNA interaction, information on local struc-
ture and dynamic features in p53 RE is important in deciphering p53-RE
recognition. Towards this goal, we report results on probing p53-RE using
site-directed spin labeling, where a site-specifically attached nitroxide radical
is monitored to obtain structural and dynamic information of the parent macro-
molecule. A nucleotide-independent labeling scheme is used to scan the nitro-
xide along RE sequences, and continuous-wave electron paramagnetic
resonance spectroscopy is used to monitor the rotational motion of the nitro-
xide, which encodes DNA structural and dynamic properties at the labeling
site. Spectral variations have been observed, with analyses of which reveals
sequence-specific local environment within the RE. This will aid in understand-
ing p53 recognition of target DNAs.
1760-Pos Board B652
Incorporation of a Rigid TOAC Spin-Label as a Non-Native Amino Acid
into a Full-Length Protein by In Vitro Translation using Amber Codon
Suppression
Amanda R. Storm, Daniel J. Mayo, Gary Lorigan, Carole Dabney-Smith.
Miami University, Oxford, OH, USA.
The development of EPR site-directed spin-labeling (SDSL) techniques has
provided a powerful avenue for the study of membrane protein structure and
dynamics. One of the advantages of EPR is the ability to obtain distance and
dynamic information about particular protein regions. The accuracy of this
information is highly dependent on the spin-label that is used. Labels with
less inherent mobility are better reporters of protein backbone dynamics and
give more sharply defined distance parameters. TOAC is a commonly used
rigid spin-label that can be integrated into the protein backbone as an amino
acid analog. However, its current use is limited to the study of small peptides
that can be only generated synthetically (solid-phase peptide synthesis). A new
biochemical approach for incorporation of non-natural amino acids during
translation using a synthetically loaded amber suppressor tRNA has success-
fully generated TOAC-labeled protein but not in sufficient yields for EPR
studies. Here, we describe the use of an amber codon suppressor tRNA
and a continuous exchange cell-free (CECF) expression system for in vitro
translation of a small thylakoid membrane protein, Tha4, containing site-
specific integration of TOAC. Further, we demonstrate successful incorpora-
tion of the TOAC-containing Tha4 into liposomes by supplying POPC vesicles
during the translation reaction. By combining new techniques in suppressor
tRNA and continuous exchange cell-free (CECF) translation, we have
developed a method for translation of full length proteins with site-directed
insertion of non-natural amino acid spin labels in yields applicable to
CW-EPR experiments.
1761-Pos Board B653
Probing the Secondary Structure of Membrane Proteins with the Pulsed
EPR ESEEM Technique
Lishan Liu.
Miami University, Oxford, OH, USA.
A novel approach is being developed to probe the secondary structure of mem-
brane proteins and peptides qualitatively utilizing the three-pulse Electron
Spin Echo Envelope Modulation (ESEEM) pulse sequence. The a-helical
M2delta subunit of the acetylcholine receptor incorporated into phospholipids
bicelles has been used as a model peptide. In order to demonstrate the practi-
cality of this method, a cysteine mutated nitroxide spin label is positioned 1, 2,
3 and 4 residues away from two types of deuterated Leu side chains (denoted
iþ1 to iþ4).
The characteristic periodicity of the a-helical structure gives rise to a unique
pattern in the ESEEM spectra. So far, this method has been successful with
2H-labeled Val and Leu. The deuterium modulation depth is significantly


Incorporation of a Rigid TOAC Spin-Label as a Non-Native Amino Acid into a Full-Length Protein by In Vitro Translation using Amber Codon Suppression 

file:///data/remote/core/dit/data/elsevier/pdf/b8d/aHR0cDovL2FwaS5lbHNldmllci5jb20vY29udGVudC9hcnRpY2xlL3BpaS9zMDAwNjM0OTUxMjAzMTUzMA==.pdf

Incorporation of a Rigid TOAC Spin-Label as a Non-Native Amino Acid into a Full-Length Protein by In Vitro Translation using Amber Codon Suppression

Abstract

Similar works

Full text

Available Versions

Elsevier - Publisher Connector

Elsevier - Publisher Connector