Publications by authors named "Alan R Penheiter"

25 Publications

  • Page 1 of 1

Identification of a pyruvate-to-lactate signature in pancreatic intraductal papillary mucinous neoplasms.

Pancreatology 2018 Jan 14;18(1):46-53. Epub 2017 Nov 14.

Department of Molecular Medicine, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA; Department of Radiology, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA. Electronic address:

Objective: We used transcriptomic profiling and immunohistochemistry (IHC) to search for a functional imaging strategy to resolve common problems with morphological imaging of cystic neoplasms and benign cystic lesions of the pancreas.

Methods: Resected pancreatic cancer (n = 21) and normal pancreas were laser-capture micro-dissected, and transcripts were quantified by RNAseq. Functional imaging targets were validated at the protein level by IHC on a pancreatic adenocarcinoma tissue microarray and a newly created tissue microarray of resected intraductal papillary mucinous neoplasms (IPMNs) and IPMN-associated adenocarcinomas.

Results: Genes encoding proteins responsible for cellular import of pyruvate, export of lactate, and conversion of pyruvate to lactate were highly upregulated in pancreatic adenocarcinoma compared to normal pancreas. Strong expression of MCT4 and LDHA was observed by IHC in >90% of adenocarcinoma specimens. In IPMNs, the pyruvate-to-lactate signature was significantly elevated in high grade dysplasia (HGD) and IPMN-associated adenocarcinoma. Additionally, cores containing HGD and/or adenocarcinoma exhibited a higher number of peri-lesional stromal cells and a significant increase in peri-lesional stromal cell staining of LDHA and MCT4. Interestingly, the pyruvate-to-lactate signature was significantly upregulated in cores containing only low grade dysplasia (LGD) from patients with histologically confirmed IPMN-associated adenocarcinoma versus LGD cores from patients with non-invasive IPMNs.

Conclusion: Our results suggest prospective studies with hyperpolarized [1-C]-pyruvate magnetic resonance spectroscopic imaging are warranted. If these IHC results translate to functional imaging findings, a positive pyruvate-to-lactate imaging signature might be a risk factor for invasion that would warrant resection of IPMNs in the absence of other worrisome features.
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http://dx.doi.org/10.1016/j.pan.2017.11.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6139027PMC
January 2018

Transcriptomic and Immunohistochemical Profiling of SLC6A14 in Pancreatic Ductal Adenocarcinoma.

Biomed Res Int 2015 27;2015:593572. Epub 2015 May 27.

Department of Molecular Medicine, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA ; Department of Radiology, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA.

We used a target-centric strategy to identify transporter proteins upregulated in pancreatic ductal adenocarcinoma (PDAC) as potential targets for a functional imaging probe to complement existing anatomical imaging approaches. We performed transcriptomic profiling (microarray and RNASeq) on histologically confirmed primary PDAC tumors and normal pancreas tissue from 33 patients, including five patients whose tumors were not visible on computed tomography. Target expression was confirmed with immunohistochemistry on tissue microarrays from 94 PDAC patients. The best imaging target identified was SLC6A14 (a neutral and basic amino acid transporter). SLC6A14 was overexpressed at the transcriptional level in all patients and expressed at the protein level in 95% of PDAC tumors. Very little is known about the role of SLC6A14 in PDAC and our results demonstrate that this target merits further investigation as a candidate transporter for functional imaging of PDAC.
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http://dx.doi.org/10.1155/2015/593572DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4461733PMC
March 2016

Interactions between beta-2 adrenoceptor gene variation, cardiovascular control and dietary sodium in healthy young adults.

J Physiol 2014 Dec 25;592(23):5221-33. Epub 2014 Sep 25.

Departments of Anaesthesiology, Physiology and Biomedical Engineering, Mayo Clinic, Rochester, MN, USA.

Dietary sodium affects function of the beta-2 adrenoceptor (ADRB2). We tested the hypothesis that haplotype variation in the ADRB2 gene would influence the cardiovascular and regional vasodilator responses to sympathoexcitatory manoeuvres following low, normal and high sodium diets, and ADRB2-mediated forearm vasodilation in the high sodium condition. Seventy-one healthy young adults were grouped by double homozygous haplotypes: Arg16+Gln27 (n = 31), the rare Gly16+Gln27 (n = 10) and Gly16+Glu27 (n = 30). Using a randomized cross-over design, subjects were studied following 5 days of controlled low, normal and high sodium with 1 month or longer between diets (and low hormone phase of the menstrual cycle). All three visits utilized ECG and finger plethysmography for haemodynamic measures, and the high sodium visit included a brachial arterial catheter for forearm vasodilator responses to isoprenaline with plethysmography. Lymphocytes were sampled for ex vivo analysis of ADRB2 density and binding conformation. We found a main effect of haplotype on ADRB2 density (P = 0.03) with the Gly16+Glu27 haplotype having the greatest density (low, normal, high sodium: 12.9 ± 0.9, 13.5 ± 0.9 and 13.6 ± 0.8 fmol mg(-1) protein, respectively) and Arg16+Gln27 having the least (9.3 ± 0.6, 10.1 ± 0.5 and 10.3 ± 0.6  fmol mg(-1) protein, respectively), but there were no sodium or haplotype effects on receptor binding conformation. In the mental stress trial, there was a main effect of haplotype on cardiac output (P = 0.04), as Arg16+Gln27 had the lowest responses. Handgrip and forearm vasodilation yielded no haplotype differences, and no correlations were present for ADRB2 density and haemodynamics. Our findings support cell-based evidence that ADRB2 haplotype influences ADRB2 protein expression independent of dietary sodium, yet the haemodynamic consequences appear modest in healthy humans.
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http://dx.doi.org/10.1113/jphysiol.2014.276469DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4262335PMC
December 2014

Neutralization capacity of measles virus H protein specific IgG determines the balance between antibody-enhanced infectivity and protection in microglial cells.

Virus Res 2013 Mar 21;172(1-2):15-23. Epub 2012 Dec 21.

Department of Molecular Medicine, Mayo Clinic, Rochester, MN 55905, USA.

Neutralizing antibodies directed against measles virus (MV) surface glycoproteins prevent viral attachment and entry through the natural receptors. H protein specific IgG can enhance MV infectivity in macrophages via Fcγ receptor (FcγR)-dependent mechanism. H-specific IgM, anti-F antibodies and complement cascade activation are protective against antibody-mediated enhancement of MV infection. However, protective role of anti-H IgG against antibody-enhanced infection is not well understood. Here we designed a set of experiments to test the protective effect of H-specific IgG against FcγR-mediated infection in microglial cells. Microglial cells are also potential target of the antibody-mediated enhancement and spread of MV infection in the central nervous system. A partially neutralizing IgG monoclonal antibody (MAb) CL55, specific for MV H protein, at 10 μg/ml enhanced MV infection in mouse microglial cells by 13-14-fold. Infection-enhancing antibody concentrations induced large multinucleated syncytia formation 48-72 h post-inoculation. We generated anti-H IgG MAb 20H6 with a strong neutralization capacity >1:80,000 at 1mg/ml concentration in MV plaque-reduction neutralization assay. In contrast to the partially protective MAb CL55, enhancement of MV infectivity by MAb 20H6 required dilutions below the 1:120 serum titer considered protective against measles infection in humans. At a concentration of 10 μg/ml MAb 20H6 exhibited a dominant protective effect and prevented MAb CL55-mediated enhancement of MV infection and virus-mediated fusion. These results indicate that neutralization capacity of the H-specific IgG determines the balance between antibody enhancement and protection against MV infection in microglial cells.
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http://dx.doi.org/10.1016/j.virusres.2012.12.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3612881PMC
March 2013

Monitoring the initial delivery of an oncolytic measles virus encoding the human sodium iodide symporter to solid tumors using contrast-enhanced computed tomography.

J Gene Med 2012 Sep-Oct;14(9-10):590-7

Department of Molecular Medicine, Mayo Clinic, Rochester, MN, USA.

Background: We aimed to determine the feasibility of monitoring viral delivery and initial distribution to solid tumors using iodinated contrast agent and micro-computed tomography (CT).

Methods: Human BxPC-3 pancreatic tumor xenografts were established in nude mice. An oncolytic measles virus with an additional transcriptional unit encoding the sodium iodide symporter (NIS), as a reporter for viral infection, was mixed with a 1:10 dilution of Omnipaque 300 (GE Healthcare, Milwaukee, WI, USA) contrast agent and injected directly into tumors. Mice were imaged with micro-CT immediately before and after injection to determine the location of contrast agent/virus mixture. Mice were imaged again on day 3 after injection with micro-single-photon emission CT/CT to determine the location of NIS-mediated (99m) TcO(4) transport.

Results: A 1:10 dilution of Omnipaque had no effect on viral infectivity or cell viability in vitro and was more than adequate for CT imaging of the intratumoral injectate distribution. The volume of tumor coverage with initial CT contrast agent and the 3-day postinfection measurement of virally infected tumor volume were significantly correlated. Additionally, regions of the tumor that did not receive contrast agent from the initial injection were largely devoid of viral infection at early time points.

Conclusions: Contrast-enhanced viral delivery enables a rapid and accurate prediction of the initial viral distribution within a solid tumor. This technique should enable real-time monitoring of viral propagation from initially infected tumor regions to adjacent tumor regions.
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http://dx.doi.org/10.1002/jgm.2670DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3930067PMC
April 2013

Development of monoclonal antibody-based immunoassays for detection of Helicobacter pylori neutrophil-activating protein.

J Immunol Methods 2012 Oct 28;384(1-2):1-9. Epub 2012 Jun 28.

Department of Molecular Medicine, Mayo Clinic, Rochester, MN 55905, USA.

Neutrophil-activating protein (NAP) is a major virulence factor expressed by Helicobacter pylori isolates associated with severe chronic gastroduodenal inflammation and peptic ulcers. NAP is one of the main protective antigens and a target for vaccine development against Helicobacter infection. In addition, NAP is a potent immune stimulator with potential application as a general vaccine adjuvant and in treatment of allergic diseases and cancer immunotherapy. NAP-specific immunoassays are needed for both H. pylori diagnostics and characterization of NAP-based vaccines and immunomodulatory preparations. We generated a panel of NAP-specific monoclonal antibodies (MAbs) by immunization of BALB/c mice with synthetic NAP peptides. The antibody reactivity against recombinant or native NAP antigen was characterized by enzyme-linked immunosorbent assay (ELISA), immunoblotting and immunofluorescence. A sensitive capture ELISA was developed using MAbs 23C8 and 16F4 (directed against different NAP epitopes) for detection of native or measles virus (MV) vector-expressed recombinant NAP in a concentration range of 4 ng/ml to 2000 ng/ml. MAb 23C8 antigen-binding depends on Tyr101 in a variable amino acid sequence of the NAP molecule, indicating the existence of antigenic variants among H. pylori strains. MAb 16F4 reacted with NAP from different H. pylori strains and was a sensitive tool for detection of small amounts of isolated NAP antigen or whole bacteria by immunoblotting or immunofluorescence. In conclusion, MAb-based immunoassays are highly specific and sensitive for detection of native NAP antigen and recombinant NAP immunostimulatory transgenes expressed by replication competent virus vectors.
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http://dx.doi.org/10.1016/j.jim.2012.06.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3691681PMC
October 2012

The sodium iodide symporter (NIS) as an imaging reporter for gene, viral, and cell-based therapies.

Curr Gene Ther 2012 Feb;12(1):33-47

Department of Molecular Medicine, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA.

Preclinical and clinical tomographic imaging systems increasingly are being utilized for non-invasive imaging of reporter gene products to reveal the distribution of molecular therapeutics within living subjects. Reporter gene and probe combinations can be employed to monitor vectors for gene, viral, and cell-based therapies. There are several reporter systems available; however, those employing radionuclides for positron emission tomography (PET) or singlephoton emission computed tomography (SPECT) offer the highest sensitivity and the greatest promise for deep tissue imaging in humans. Within the category of radionuclide reporters, the thyroidal sodium iodide symporter (NIS) has emerged as one of the most promising for preclinical and translational research. NIS has been incorporated into a remarkable variety of viral and non-viral vectors in which its functionality is conveniently determined by in vitro iodide uptake assays prior to live animal imaging. This review on the NIS reporter will focus on 1) differences between endogenous NIS and heterologously-expressed NIS, 2) qualitative or comparative use of NIS as an imaging reporter in preclinical and translational gene therapy, oncolytic viral therapy, and cell trafficking research, and 3) use of NIS as an absolute quantitative reporter.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3367315PMC
http://dx.doi.org/10.2174/156652312799789235DOI Listing
February 2012

Smooth muscle myosin light chain kinase efficiently phosphorylates serine 15 of cardiac myosin regulatory light chain.

Biochem Biophys Res Commun 2011 Dec 19;416(3-4):367-71. Epub 2011 Nov 19.

Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN 55905, USA.

Specific phosphorylation of the human ventricular cardiac myosin regulatory light chain (MYL2) modifies the protein at S15. This modification affects MYL2 secondary structure and modulates the Ca(2+) sensitivity of contraction in cardiac tissue. Smooth muscle myosin light chain kinase (smMLCK) is a ubiquitous kinase prevalent in uterus and present in other contracting tissues including cardiac muscle. The recombinant 130 kDa (short) smMLCK phosphorylated S15 in MYL2 in vitro. Specific modification of S15 was verified using the direct detection of the phospho group on S15 with mass spectrometry. SmMLCK also specifically phosphorylated myosin regulatory light chain S15 in porcine ventricular myosin and chicken gizzard smooth muscle myosin (S20 in smooth muscle) but failed to phosphorylate the myosin regulatory light chain in rabbit skeletal myosin. Phosphorylation kinetics, measured using a novel fluorescence method eliminating the use of radioactive isotopes, indicates similar Michaelis-Menten V(max) and K(M) for regulatory light chain S15 phosphorylation rates in MYL2, porcine ventricular myosin, and chicken gizzard myosin. These data demonstrate that smMLCK is a specific and efficient kinase for the in vitro phosphorylation of MYL2, cardiac, and smooth muscle myosin. Whether smMLCK plays a role in cardiac muscle regulation or response to a disease causing stimulus is unclear but it should be considered a potentially significant kinase in cardiac tissue on the basis of its specificity, kinetics, and tissue expression.
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http://dx.doi.org/10.1016/j.bbrc.2011.11.044DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3242870PMC
December 2011

Sodium iodide symporter (NIS)-mediated radiovirotherapy for pancreatic cancer.

AJR Am J Roentgenol 2010 Aug;195(2):341-9

Department of Molecular Medicine, Mayo Clinic, Rochester, MN 55905, USA.

Objective: We have previously shown the therapeutic efficacy of an engineered oncolytic measles virus expressing the sodium iodide symporter reporter gene (MV-NIS) in mice with human pancreatic cancer xenografts. The goal of this study was to determine the synergy between MV-NIS-induced oncolysis and NIS-mediated (131)I radiotherapy in this tumor model.

Materials And Methods: Subcutaneous human BxPC-3 pancreatic tumors were injected twice with MV-NIS. Viral infection, NIS expression, and intratumoral iodide uptake were quantitated with (123)I micro-SPECT/CT. Mice with MV-NIS-infected tumors were treated with 0, 37, or 74 MBq (131)I and monitored for tumor progression and survival. Additional studies were performed with stable NIS-expressing tumors (BxPC-3-NIS) treated with 0, 3.7, 18.5, 37, or 74 MBq of (131)I.

Results: Mice treated with intratumoral MV-NIS exhibited significant tumor growth delay (p < 0.01) and prolonged survival (p = 0.02) compared with untreated mice. Synergy between MV-NIS-induced oncolysis and NIS-mediated (131)I ablation was not seen; however, a significant correlation was observed between NIS-mediated intratumoral iodide localization (% ID/g) and peak tumor volume reduction (p = 0.04) with combination MV-NIS and (131)I therapy. Stably transduced NIS-expressing BxPC-3 tumors exhibited rapid regression with > or = 18.5 MBq (131)I.

Conclusion: Delivery of (131)I radiotherapy to NIS-expressing tumors can be optimized using micro-SPECT/CT imaging guidance. Significant hurdles exist for NIS as a therapeutic gene for combined radiovirotherapy in this human pancreatic cancer model. The lack of synergy observed with MV-NIS and (131)I in this model was not due to a lack of radiosensitivity but rather to a nonuniform intratumoral distribution of MV-NIS infection.
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http://dx.doi.org/10.2214/AJR.09.3672DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3117397PMC
August 2010

beta2-Adrenoceptor gene variation and systemic vasodilatation during ganglionic blockade.

J Physiol 2010 Jul 2;588(Pt 14):2669-78. Epub 2010 Jun 2.

Department of Anaesthesiology, Internal Medicine, Mayo Clinic, Rochester, MN 55905, USA.

Regional infusions of beta(2)-adrenoceptor (ADRB2) agonist have generally shown that individuals homozygous for Gly16 produces greater vasodilatation than those homozygous for Arg16. Systemic infusions have shown an opposite effect on systemic vascular resistance (SVR), possibly confounded by baroreflexes or interactions between single nucleotide polymorphism (SNP) positions 16 and 27. We tested the hypothesis that ADRB2 gene variation would influence the SVR response to ADRB2 agonist terbutaline (Terb) during ganglionic blockade. Forty healthy young adults were recruited according to the double homozygous haplotypes: Arg16 + Gln27 (n = 13), the rare Gly16 + Gln27 (n = 6), and Gly16 + Glu27 (n = 21). Arterial pressure was measured by brachial arterial catheter, and cardiac output by acetylene breathing. Lymphocytes were sampled for ex vivo analysis of ADRB2 density and binding conformation. Following baroreflex ablation with trimethaphan (3-7 mg min(1)), continuous phenylephrine was titrated to restore blood pressure to baseline. Terb was infused i.v. at 33 and 67 ng kg(1) min(1) for 15 min/dose. There was partial evidence to suggest a main effect of haplotype on the change in SVR (P = 0.06). For SNP position 16, the highest dose of Terb produced lower SVR in Gly16 (mean +/- s.e.m.: 7.5 +/- 0.4) vs. Arg16 (8.9 +/- 0.7 units; P = 0.03). Lymphocyte ADRB2 binding conformation was similar but receptor density was greater in Gly16 vs. Arg16 (P = 0.05). We conclude that during ganglionic blockade, the SVR response to systemic ADRB2 agonist is suggestive of augmented ADRB2 function in Gly16 + Glu27 homozygotes, with greater influence from Gly16, providing further evidence that ADRB2 gene variation influences vasodilatation.
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http://dx.doi.org/10.1113/jphysiol.2010.190058DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2916996PMC
July 2010

The myosin C-loop is an allosteric actin contact sensor in actomyosin.

Biochemistry 2009 Jun;48(23):5263-75

Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, Minnesota 55905, USA.

Actin and myosin form the molecular motor in muscle. Myosin is the enzyme performing ATP hydrolysis under the allosteric control of actin such that actin binding initiates product release and force generation in the myosin power stroke. Non-equilibrium Monte Carlo molecular dynamics simulation of the power stroke suggested that a structured surface loop on myosin, the C-loop, is the actin contact sensor initiating actin activation of the myosin ATPase. Previous experimental work demonstrated C-loop binds actin and established the forward and reverse allosteric link between the C-loop and the myosin active site. Here, smooth muscle heavy meromyosin C-loop chimeras were constructed with skeletal (sCl) and cardiac (cCl) myosin C-loops substituted for the native sequence. In both cases, actin-activated ATPase inhibition is indicated mainly by the lower V(max). In vitro motility was also inhibited in the chimeras. Motility data were collected as a function of myosin surface density, with unregulated actin, and with skeletal and cardiac isoforms of tropomyosin-bound actin for the wild type, cCl, and sCl. Slow and fast subpopulations of myosin velocities in the wild-type species were discovered and represent geometrically unfavorable and favorable actomyosin interactions, respectively. Unfavorable interactions are detected at all surface densities tested. Favorable interactions are more probable at higher myosin surface densities. Cardiac tropomyosin-bound actin promotes the favorable actomyosin interactions by lowering the inhibiting geometrical constraint barriers with a structural effect on actin. Neither higher surface density nor cardiac tropomyosin-bound actin can accelerate motility velocity in cCl or sCl, suggesting the element initiating maximal myosin activation by actin resides in the C-loop.
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http://dx.doi.org/10.1021/bi900584qDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2759872PMC
June 2009

An unusual transduction pathway in human tonic smooth muscle myosin.

Biophys J 2007 Nov 17;93(10):3555-66. Epub 2007 Aug 17.

Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, Minnesota, USA.

The motor protein myosin binds actin and ATP, producing work by causing relative translation of the proteins while transducing ATP free energy. Smooth muscle myosin has one of four heavy chains encoded by the MYH11 gene that differ at the C-terminus and in the active site for ATPase due to alternate splicing. A seven-amino-acid active site insert in phasic muscle myosin is absent from the tonic isoform. Fluorescence increase in the nucleotide sensitive tryptophan (NST) accompanies nucleotide binding and hydrolysis in several myosin isoforms implying it results from a common origin within the motor. A wild-type tonic myosin (smA) construct of the enzymatic head domain (subfragment 1 or S1) has seven tryptophan residues and nucleotide-induced fluorescence enhancement like other myosins. Three smA mutants probe the molecular basis for the fluorescence enhancement. W506+ contains one tryptophan at position 506 homologous to the NST in other myosins. W506F has the native tryptophans except phenylalanine replaces W506, and W506+(Y499F) is W506+ with phenylalanine replacing Y499. W506+ lacks nucleotide-induced fluorescence enhancement probably eliminating W506 as the NST. W506F has impaired ATPase activity but retains nucleotide-induced fluorescence enhancement. Y499F replacement in W506+ partially rescues nucleotide sensitivity demonstrating the role of Y499 as an NST facilitator. The exceptional response of W506 to active site conformation opens the possibility that phasic and tonic isoforms differ in how influences from active site ATPase propagate through the protein network.
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http://dx.doi.org/10.1529/biophysj.106.100818DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2072059PMC
November 2007

Cleavage of the plasma membrane Ca+ATPase during apoptosis.

Ann N Y Acad Sci 2007 Mar;1099:440-50

Membrane Research Group of the Hungarian Academy of Sciences, Budapest H-1051, Hungary.

Maintenance of Ca2+ homeostasis is essential for normal cellular function and survival. Recent evidences suggest that Ca2+ is also an important player of apoptosis. We demonstrated that the plasma membrane Ca2+ ATPase (PMCA) isoform 4b, a key element of cellular Ca2+ homeostasis, was cleaved by caspase-3 during the course of apoptosis. This cleavage of PMCA removed the entire regulatory region from the C terminus, leaving behind a 120-kDa catalytic fragment. Since loss of PMCA activity could lead to intracellular Ca2+ overload and consequently necrotic cell death, an important question is whether the apoptotic fragment of PMCA retains full activity or it is inactivated. To address this question, we constructed a C-terminally truncated mutant that corresponded to the caspase-3 fragment of PMCA4b and showed that it was fully and constitutively active. This mutant was targeted properly to the plasma membrane when it was expressed stably or transiently in several different cell lines. We followed truncation of PMCA during apoptosis induced by mitochondrial or receptor-mediated pathways and found that a similar fragment of 120 kDa was formed and remained intact for several hours after treatment. We have also demonstrated that the caspase-3 cleavage site is an important structural element of PMCA and found that the accessibility of the caspase-3 site depended strongly on the conformational state of the protein.
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http://dx.doi.org/10.1196/annals.1387.003DOI Listing
March 2007

H(2)O(2)-induced kinetic and chemical modifications of smooth muscle myosin: correlation to effects of H(2)O(2) on airway smooth muscle.

J Biol Chem 2007 Feb 22;282(7):4336-4344. Epub 2006 Nov 22.

Department of Biochemistry and Molecular Biology, University of Nevada School of Medicine, Reno, Nevada 89557. Electronic address:

The effect of H(2)O(2) on smooth muscle heavy meromyosin (HMM) and subfragment 1 (S1) was examined. The number of molecules that retained the ability to bind ATP and the actinactivated rate of P(i) release were measured by single-turnover kinetics. H(2)O(2) treatment caused a decrease in HMM regulation from 800- to 27-fold. For unphosphorylated and phosphorylated heavy meromyosin and for S1, approximately 50% of the molecules lost the ability to bind to ATP. H(2)O(2) treatment in the presence of EDTA protected against ATPase inactivation and against the loss of total ATP binding. Inactivation of S1 versus time correlated to a loss of reactive thiols. Treatment of H(2)O(2)-inactivated phosphorylated HMM or S1 with dithiothreitol partially reactivated the ATPase but had no effect on total ATP binding. H(2)O(2)-inactivated S1 contained a prominent cross-link between the N-terminal 65-kDa and C-terminal 26-kDa heavy chain regions. Mass spectral studies revealed that at least seven thiols in the heavy chain and the essential light chain were oxidized to cysteic acid. In thiophosphorylated porcine tracheal muscle strips at pCa 9 + 2.1 mM ATP, H(2)O(2) caused a approximately 50% decrease in the amplitude but did not alter the rate of force generation, suggesting that H(2)O(2) directly affects the force generating complex. Dithiothreitol treatment reversed the H(2)O(2) inhibition of the maximal force by approximately 50%. These data, when compared with the in vitro kinetic data, are consistent with a H(2)O(2)-induced loss of functional myosin heads in the muscle.
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http://dx.doi.org/10.1074/jbc.M609499200DOI Listing
February 2007

Prediction of volatile anesthetic binding sites in proteins.

Biophys J 2006 Nov 28;91(9):3405-14. Epub 2006 Jul 28.

Departments of Anesthesiology and Molecular Pharmacology and Experimental Therapeutics and Biochemistry and Molecular Biology, Mayo College of Medicine, Rochester, Minnesota, USA.

Computational methods designed to predict and visualize ligand protein binding interactions were used to characterize volatile anesthetic (VA) binding sites and unoccupied pockets within the known structures of VAs bound to serum albumin, luciferase, and apoferritin. We found that both the number of protein atoms and methyl hydrogen, which are within approximately 8 A of a potential ligand binding site, are significantly greater in protein pockets where VAs bind. This computational approach was applied to structures of calmodulin (CaM), which have not been determined in complex with a VA. It predicted that VAs bind to [Ca(2+)](4)-CaM, but not to apo-CaM, which we confirmed with isothermal titration calorimetry. The VA binding sites predicted for the structures of [Ca(2+)](4)-CaM are located in hydrophobic pockets that form when the Ca(2+) binding sites in CaM are saturated. The binding of VAs to these hydrophobic pockets is supported by evidence that halothane predominantly makes contact with aliphatic resonances in [Ca(2+)](4)-CaM (nuclear Overhauser effect) and increases the Ca(2+) affinity of CaM (fluorescence spectroscopy). Our computational analysis and experiments indicate that binding of VA to proteins is consistent with the hydrophobic effect and the Meyer-Overton rule.
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http://dx.doi.org/10.1529/biophysj.106.082586DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1614498PMC
November 2006

Differential effects of volatile anesthetics on M3 muscarinic receptor coupling to the Galphaq heterotrimeric G protein.

Anesthesiology 2006 Aug;105(2):313-24

Department of Anesthesiology, Mayo Foundation, Rochester, Minnesota 55905, USA.

Background: Halothane inhibits airway smooth muscle contraction in part by inhibiting the functional coupling between muscarinic receptors and one of its cognate heterotrimeric G proteins, Galphaq. Based on previous studies indicating a more potent effect of halothane and sevoflurane on airway smooth muscle contraction compared with isoflurane, the current study hypothesized that at anesthetic concentrations of 2 minimum alveolar concentration (MAC) or less, halothane and sevoflurane but not isoflurane inhibit acetylcholine-promoted Galphaq guanosine nucleotide exchange.

Methods: Galphaq guanosine nucleotide exchange was measured in crude membranes prepared from COS-7 cells transiently coexpressing the human M3 muscarinic receptor and human Galphaq. A radioactive, nonhydrolyzable analog of guanosine-5'-triphosphate, [35S]GTPgammaS, was used as a reporter for nucleotide exchange at Galphaq.

Results: Acetylcholine caused a concentration-dependent increase in Galphaq [35S]GTPgammaS-GDP exchange. Neither anesthetic affected constitutive Galphaq [35S]GTPgammaS-GDP exchange in the absence of acetylcholine. Conversely, each anesthetic caused a concentration-dependent and reversible inhibition of Galphaq [35S]GTPgammaS-GDP exchange when promoted by acetylcholine. At concentrations of 3 MAC or less, the effect of halothane and sevoflurane were significantly greater than that of isoflurane, with only a minimal inhibition by isoflurane observed at 2 MAC.

Conclusion: The differential effects of volatile anesthetics on acetylcholine-promoted guanosine nucleotide exchange at Galphaq are consistent with the apparent more potent direct effect of halothane and sevoflurane compared with isoflurane on muscarinic receptor-mediated contraction of isolated airway smooth muscle. These differential effects also suggest a mode of anesthetic action that could be due to anesthetic-protein interactions and not simply anesthetic accumulation in the lipid membrane.
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http://dx.doi.org/10.1097/00000542-200608000-00014DOI Listing
August 2006

Halothane does not inhibit the functional coupling between the beta2-adrenergic receptor and the Galphas heterotrimeric G protein.

Anesthesiology 2006 Apr;104(4):754-62

Department of Anesthesiology, Mayo Foundation, Rochester, Minnesota 55905, USA.

Background: This study investigated whether halothane affects the functional coupling between the beta2 adrenergic receptor and the alpha subunit of its cognate stimulatory heterotrimeric guanosine-5'-triphosphate (GTP)-binding protein (Galphas). The authors hypothesized that halothane does not affect isoproterenol-promoted guanosine nucleotide exchange at Galphas and hence would not affect isoproterenol-induced relaxation of airway smooth muscle.

Methods: Halothane effects on isoproterenol-induced inhibition of calcium sensitivity were measured in permeabilized porcine airway smooth muscle. Galphas nucleotide exchange was measured in crude membranes prepared from COS-7 cells transfected to transiently coexpress the human beta1 or beta2 receptor each with human short Galphas. A radioactive, nonhydrolyzable analog of GTP, [S]GTPgammaS, was used as the reporter for nucleotide exchange at Galphas.

Results: Halothane (0.75 mm, approximately 2.8 minimum alveolar concentration [MAC] in pigs) did not affect isoproterenol-induced inhibition of calcium sensitivity. Isoproterenol caused a time- and concentration-dependent increase in Galphas nucleotide exchange. Halothane, even at concentrations of 1.5 mm (approximately 5.6 MAC), had no effect on basal Galphas nucleotide exchange in the absence of isoproterenol, whereas halothane inhibited isoproterenol-promoted Galphas nucleotide exchange in both the beta1-Galphas and beta2-Galphas expressing membranes. However, the effect was significantly greater on beta1-Galphas coupling compared with beta2-Galphas coupling, with no effect on beta2-Galphas coupling at 2.8 MAC halothane.

Conclusion: Halothane does not inhibit the biochemical coupling between the beta2 receptor and Galphas and hence does not affect the inhibition of calcium sensitivity induced by isoproterenol. Therefore, halothane should not affect the efficacy of beta2 agonists, as suggested by studies of in vivo animal models of asthma.
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http://dx.doi.org/10.1097/00000542-200604000-00020DOI Listing
April 2006

Effect of halothane on galphai-3 and its coupling to the M2 muscarinic receptor.

Anesthesiology 2005 Nov;103(5):1015-25

Department of Anesthesiology, Mayo Foundation, Rochester, Minnesota 55905, USA.

Background: Halothane is an effective bronchodilator and inhibits airway smooth muscle contraction in part by inhibiting intracellular signaling pathways activated by the M2 muscarinic receptor and its cognate inhibitory heterotrimeric guanosine-5'-triphosphate (GTP)-binding protein (G protein), Gi. This study hypothesized that halothane inhibits nucleotide exchange at the alpha isoform-3 subunit of Gi (Galphai-3), but only when regulated by the M2 muscarinic receptor.

Methods: GTP hydrolysis by Galphai-3 and the Galphai-3beta1gamma2HF heterotrimer expressed in Spodoptera frugiperda cells was measured using a phosphohydrolase assay with [gammaPi]-labeled GTP. Anesthetic binding to Galphai-3 was measured by saturation transfer difference nuclear magnetic resonance spectroscopy. Galphai-3 nucleotide exchange was measured in crude membranes prepared from COS-7 cells transiently coexpressing the M2 muscarinic receptor and Galphai-3. A radioactive analog of GTP, [S]GTPgammaS, was used as a reporter for Galphai-3 nucleotide exchange.

Results: Although spectroscopy demonstrated halothane binding to Galphai-3, this binding had no effect on [gammaPi]-labeled GTP hydrolysis by the Galphai-3beta1gamma2HF heterotrimer expressed in Spodoptera frugiperda cells, nor basal Galphai-3 nucleotide exchange measured in crude membranes when the muscarinic receptor agonist acetylcholine was omitted from the assay. Conversely, halothane caused a concentration-dependent inhibition of Galphai-3 nucleotide exchange with acetylcholine included in the assay.

Conclusion: These data indicate that despite halothane binding to Galphai-3, halothane has no direct inhibitory effect on the intrinsic activity of the Galphai-3beta1gamma2HF heterotrimer but inhibits M2 muscarinic receptor regulation of the heterotrimer. This novel effect is consistent with the ability of halothane to inhibit airway smooth muscle contraction and bronchoconstriction induced by acetylcholine.
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http://dx.doi.org/10.1097/00000542-200511000-00016DOI Listing
November 2005

The caspase-3 cleavage product of the plasma membrane Ca2+-ATPase 4b is activated and appropriately targeted.

Biochem J 2005 Nov;391(Pt 3):687-92

Membrane Research Group of the Hungarian Academy of Sciences, Nádor u.7., H-1051, Budapest, Hungary.

The calmodulin-activated transporter hPMCA4 (human plasma membrane Ca2+-ATPase isoform 4) is a target for cleavage by caspase-3 during apoptosis. We have demonstrated that caspase-3 generates a 120 kDa fragment of this pump which lacks the complete autoinhibitory sequence [Paszty, Verma, Padanyi, Filoteo, Penniston and Enyedi (2002) J. Biol. Chem. 277, 6822-6829]. In the present study we analysed further the characteristics of the fragment of hPMCA4b produced by caspase-3. We did this by overexpressing the caspase-3 cleavage product of hPMCA4b in COS-7 and MDCKII (Madin-Darby canine kidney II) cells. This technique made it possible to clearly define the properties of this fragment, and we showed that it is constitutively active, as it forms a phosphoenzyme intermediate and has high Ca2+ transport activity in the absence of calmodulin. When this fragment of hPMCA4b was stably expressed in MDCKII cell clones, it was targeted without degradation to the basolateral plasma membrane. In summary, our studies emphasize that the caspase-3 cleavage product of hPMCA4b is constitutively active, and that the C-terminus is not required for proper targeting of hPMCA4b to the plasma membrane. Also, for the first time, we have generated cell clones that stably express a constitutively active PMCA.
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http://dx.doi.org/10.1042/BJ20051012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1276970PMC
November 2005

Modification of human hearing loss by plasma-membrane calcium pump PMCA2.

N Engl J Med 2005 Apr;352(15):1557-64

Section on Human Genetics, National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Rockville, Md 20850, USA.

Five adult siblings presented with autosomal recessive sensorineural hearing loss: two had high-frequency loss, whereas the other three had severe-to-profound loss affecting all frequencies. Genetic evaluation revealed that a homozygous mutation in CDH23 (which encodes cadherin 23) caused the hearing loss in all five siblings and that a heterozygous, hypofunctional variant (V586M) in plasma-membrane calcium pump PMCA2, which is encoded by ATP2B2, was associated with increased loss in the three severely affected siblings. V586M was detected in two unrelated persons with increased sensorineural hearing loss, in the other caused by a mutation in MYO6 (which encodes myosin VI) in one and by noise exposure, suggesting that this variant may modify the severity of sensorineural hearing loss caused by a variety of factors.
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http://dx.doi.org/10.1056/NEJMoa043899DOI Listing
April 2005

Kinetic analysis of the calmodulin-binding region of the plasma membrane calcium pump isoform 4b.

Biochemistry 2005 Feb;44(6):2009-20

Department of Biochemistry and Molecular Biology, Mayo Clinic, 200 First Street SW, Rochester, Minnesota 55901, USA.

The sequence L(1086)RRGQILWFRGLNRIQTQIKVVKAFHSS(1113) (peptide C28) is responsible for calmodulin binding to PMCA4b. In this work, peptides following the above sequence were progressively shortened either at the N-terminus (C28NDelta3, C28NDelta5, or C28NDelta6) or at the C-terminus (C20, C22, C23, and C25). Competitive inhibition of PMCA activity was used to measure apparent dissociation constants of the complexes between calmodulin and C28 or progressively shortened peptides. Additionally, equilibrium titrations were used to measure the apparent dissociation constants of the various peptides with TA-calmodulin by changes in TA-calmodulin fluorescence and Trp fluorescence of the peptides. At the N-terminus, deletion of five residues did not change calmodulin affinity, but deletion of six residues resulted in a 5-fold decrease in affinity. There were no major differences in the time course of TA-CaM binding, but C28NDelta6 exhibited a different time course of Trp fluorescence change. At the C-terminus, deletion of five residues (C23) or more resulted in a net increase in fluorescence of TA-CaM upon binding, while longer peptides (C25 and C28) produced both a transient increase and a net decrease in the fluorescence of TA-CaM. Global regression analysis revealed that binding of TA-CaM to the C23 peptide could be fit by a two-step model, while longer peptides required three-step models for adequate fitting. TA-calmodulin dissociated rapidly from C23, C22, and C20, resulting in a marked increase in apparent K(d). Thus, the sequence I(1091)LWFRGLNRIQTQIKVVKAF(1110) (C25NDelta5) is required to reproduce the calmodulin-binding properties of C28. When F(1110) was replaced by A, the TA-calmodulin association and dissociation kinetics resembled C23 kinetics, but changing V(1107) to A produced a smaller effect, suggesting that F(1110), rather than V(1107), is the main anchor for the N-terminal lobe of calmodulin in PMCA4b.
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http://dx.doi.org/10.1021/bi0488552DOI Listing
February 2005

A model for the activation of plasma membrane calcium pump isoform 4b by calmodulin.

Biochemistry 2003 Oct;42(41):12115-24

Department of Biochemistry and Molecular Biology, Mayo Clinic, 200 First Street SW, Rochester, Minnesota 55901, USA.

Overexpression of the plasma membrane calcium pump (PMCA) isoform 4b by means of the baculovirus system enabled us, for the first time, to study the kinetics of calmodulin binding to this pump. This was done by stopped-flow fluorescence measurements using 2-chloro-(amino-Lys(75))-[6-[4-(N,N-diethylamino)phenyl]-1,3,5-triazin-4-yl]calmodulin (TA-calmodulin). Upon mixing with PMCA, the fluorescence of TA-calmodulin changed along a biphasic curve: a rapid and small increase in fluorescence was followed by a slow and large decrease that lasted about 100 s. The experiment was done at several PMCA concentrations. Global fitting nonlinear regression analysis of these results led to a model in which PMCA is present in two forms: a closed conformation and an open conformation. Calmodulin reacts with both conformations but reacts faster and with higher affinity for the open conformation. Measurements of the ATPase activity of PMCA under similar conditions revealed that the open form has higher ATPase activity than the closed one. Contrasting with the reaction with the whole pump, TA-calmodulin reacted rapidly (in about 2 s) with a calmodulin-binding peptide made after the sequence of the calmodulin-binding domain of PMCA (C28). Results of TA-calmodulin binding to C28 are explained by a simpler model, in which only an open conformation exists.
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http://dx.doi.org/10.1021/bi027098+DOI Listing
October 2003

Intramolecular interactions of the regulatory region with the catalytic core in the plasma membrane calcium pump.

J Biol Chem 2003 Sep 26;278(37):35798-804. Epub 2003 Jun 26.

National Medical Center, Diószegi utca 64, H-1113 Budapest, Hungary.

The access of three proteases to their sites of cleavage was used as a measure of regulatory interactions in the plasma membrane Ca2+ pump isoform 4b (PMCA4b). When the proteases could not cut at their sites in the C-terminal regulatory region, the interaction was judged to be tight. This was the case in the absence of Ca2+, when chymotrypsin and caspase cut PMCA only very slowly. Ca2+ accelerated the fragmentation, but the digestion remained incomplete. In the presence of Ca2+ plus calmodulin, the digestion became nearly complete in all cases, indicating a more flexible conformation of the carboxyl terminus in the fully activated state. The acceleration of proteolysis by Ca2+ or Ca2+ plus calmodulin occurred equally at the caspase site upstream of the calmodulin-binding domain and the chymotrypsin and calpain sites downstream of that domain. Replacing Trp1093 (a key residue within the calmodulin-binding domain) with alanine had a much more specific effect, because it exposed only proteolytic sites within the calmodulin-binding domain that had previously been shielded in the native protein. At these sites, both calpain and chymotrypsin cut the Trp1093 --> Ala mutant in the absence of calmodulin. These data indicate that, in the auto-inhibited conformation, the calmodulin-binding/auto-inhibitory sequence and the regions both upstream and downstream are in close contact with the catalytic core. Trp1093 plays an essential role not only in stabilizing the Ca2+-calmodulin/calmodulin-binding domain complex but also in the formation or stability of the inhibitory conformation of that domain when it interacts with the catalytic core of PMCA4b.
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http://dx.doi.org/10.1074/jbc.M305794200DOI Listing
September 2003

Asp1080 upstream of the calmodulin-binding domain is critical for autoinhibition of hPMCA4b.

J Biol Chem 2002 Sep 26;277(39):36146-51. Epub 2002 Jul 26.

National Medical Center, Institute of Haematology and Immunology, Diószegi u. 64, H-1113 Budapest, Hungary.

The role of the plasma membrane Ca(2+) pump (PMCA) is to remove excess Ca(2+) from the cytosol to maintain low intracellular Ca(2+) levels. Asp(1080) lies within an acidic sequence between the C-terminal inhibitory region and the catalytic core of PMCAs and is part of the caspase-3 recognition site of isoform 4b. Caspase-3 cuts immediately after this residue and activates the pump by removing the inhibitory region (Pászty, K., Verma, A. K., Padányi, R., Filoteo, A. G., Penniston, J. T., and Enyedi, A. (2002) J. Biol. Chem. 277, 6822-6829). Asp(1080) had not been believed to have any other role, but here we show that it also plays a critical role in the autoinhibition and calmodulin activation of PMCA4b. Site-specific mutation of Asp(1080) to Asn, Ala, or Lys in PMCA4b resulted in a substantial increase in the basal activity in the absence of calmodulin. All Asp(1080) mutants exhibited an increased affinity for calmodulin because of an increase in the rate of activation by calmodulin. This rate was higher when the inhibition was weaker, showing that a strong inhibitory interaction slows the activation rate. In contrast, mutating the nearby Asp(1077) had no effect on basal activity or calmodulin activation. We propose that the conserved Asp(1080), even though it is neither in the regulatory domain nor in the catalytic core, plays an essential role in inhibition by stabilizing the inhibited state of the enzyme.
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http://dx.doi.org/10.1074/jbc.M205457200DOI Listing
September 2002

Tryptophan 1093 is largely responsible for the slow off rate of calmodulin from plasma membrane Ca2+ pump 4b.

J Biol Chem 2002 May 8;277(20):17728-32. Epub 2002 Mar 8.

Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota 55905, USA.

Tryptophan 1093 resides in the 28-residue calmodulin-binding/autoinhibitory domain of the plasma membrane Ca(2+) pump (PMCA). Previous studies with the isolated calmodulin-binding/autoinhibitory peptide from PMCA have shown that mutations of the tryptophan residue decrease the affinity of the peptide for calmodulin and its affinity as an inhibitor of proteolytically activated pump. In this study, the PMCA mutation in which tryptophan 1093 is converted to alanine (W1093A) was constructed in the full-length PMCA isoform 4b. The mutant pump was expressed in COS cells, and its steady state and pre-steady state kinetic properties were examined. The W1093A pump exhibited an increased basal activity in the absence of calmodulin, so the activation was approximately 2-fold (it is 10-fold in the wild type). The W1093A mutation also lowered the steady state affinity for calmodulin from K(0.5) of 9 nm for wild type to 144 nm (assayed at 700 nm free Ca(2+)). Pre-steady state measurements of the rate of activation by Ca(2+)-calmodulin revealed that the W1093A mutant responded 2.5-fold faster to calmodulin. In contrast to these relatively modest effects, the half-time of inactivation of the mutant was reduced by more than 2 orders of magnitude from 41 min to 7 s. We conclude that tryptophan 1093 does not play a substantial role in Ca(2+)-calmodulin recognition; rather it functions primarily to slow the inactivation of the calmodulin-activated pump.
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http://dx.doi.org/10.1074/jbc.M111608200DOI Listing
May 2002