Overexpression of caveolin-1 results in increased plasma membrane trageting of phosphofructokinase: The structural basis for a membrane associated metabolic compartment

Abstract only availablePrevious work from our laboratory has shown that glycolysis and gluconeogenesis occur in separate compartments within the vascular smooth muscle cell ( VSM ) and that their intermediates do not mix freely in the cytoplasm. We have previously found that the glycolytic -specific enzyme phosphofructokinase ( PFK ) appeared to colocalize with the fairly ubiquitous plasma membrane protein caveolin -1 ( CAV - 1 ), consistent with a role for CAV - 1 as an anchor for glycolysis to the plasma membrane. We hypothesized that CAV - 1 serves as a scaffolding protein for PFK and may play a role for the organization of cell metabolism. To test this hypothesis , we over -expressed the CAV - 1 gene in cultured A7r5 ( rat aorta VSM ) cells by transfection with a CAV - 1 coding plasmid. Immunoflorescence and confocal microscopy were used to study the distribution of PFK and CAV - 1 in the transfected cells. Areas of Interest ( AOI ) were analyzed in a central z -plane across the cell transvesing the perinuclear region. To quantify any shift in PFK localization resulting from CAV - 1 over -expression , we calculated a periphery to center ( PC ) index by taking the average of the two outer AOIs from each membrane region and dividing by the central one or two AOIs. We found that the PC was 2. 2 5 + / - 0. 8 5 ( mean + / - SEM, N = 5 ) for transfected cells and was 0. 4 7 + / - 0. 1 6 for control cells. These results demonstrate that CAV - 1 creates binding sites for PFK that may be of higher affinity than those localized in the cytoplasm ( such as microtubules and the actin cytoskeleton ). We therefore conclude that CAV - 1 functions as a scaffolding protein for PFK and that this may contribute to the elucidation of the basis for carbohydrate compartmentation to the plasma membrane in VSM. Support provided by NIH 60668 (to Christopher D. Hardin) and Life Sciences Undergraduate Research Opportunity Program (LS UROP).Life Sciences Undergraduate Research Opportunity Progra

Mutations to the caveolin scaffolding domain reduces Caveolin-1 targeting of glycolytic enzymes to lymphocyte membranes [abstract]

Abstract only availablePreviously, we found caveolin (CAV-1) expressed by transfection in cultured lymphocytes induced caveolae formation and targeted the glycolytic enzyme phosphofructokinase (PFK) to the membrane. We also found CAV-1 targets other glycolytic enzymes such as aldolase (ALD)and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to the plasma membrane in the CAV-1 transfected lymphocytes. Here we hypothesized that if a mutant CAV-1 (which has essential aromatic residues in the caveolin scaffolding domain (CSD) mutated) is expressed in the lymphocyte then colocalization of the glycolytic enzyme PFK with CAV-1 will be reduced. We tested this hypothesis by comparing the colocalization of CAV-1 with the glycolytic enzymes PFK, ALD and GAPDH in lymphocytes which expressed either a wild type CAV-1 (WT) or a mutant CAV-1 which had either one mutation (SM) or two mutations (DM) in the CSD. Colocalization analysis by confocal microscopy of cells immunoassayed for CAV-1 and ALD was 76.59% in lymphocytes transfected with CAV-1 WT, 23.96% in lymphocytes transfected with CAV-1 SM, and 58.74% in the lymphocytes transfected with CAV-1 DM. Analysis of colocalization of the enzymes PFK, GAPDH, and ALD with CAV-1 averaged 65.17% for the CAV-1 WT cells, 49.29% for the CAV-1 SM cells and 50.81% for the CAV-1 DM cells. The shift in distribution of glycolytic enzymes and CAV-1 in the CAV-1 WT, the CAV-1 SM or DM CAV-1 types indicates that a single mutation to the CSD reduces membrane targeting of glycolytic enzymes, and two mutations in the CSD produces retention of CAV-1 in the cytosol. These results suggest that an intact CSD domain is essential to the CAV-1 targeting of glycolytic enzymes to the membrane

High Redshift Supernova Rates

We use a sample of 42 supernovae detected with the Advanced Camera for Surveys on-board the Hubble Space Telescope as part of the Great Observatories Origins Deep Survey to measure the rate of core collapse supernovae to z~0.7 and type Ia supernovae to z~1.6. This significantly increases the redshift range where supernova rates have been estimated from observations. The rate of core collapse supernovae can be used as an independent probe of the cosmic star formation rate. Based on the observations of 17 core collapse supernovae, we measure an increase in the core collapse supernova rate by a factor of 1.6 in the range 0.3<z<0.7, and an overall increase by a factor of 7 to z~0.7 in comparison to the local core collapse supernova rate. The increase in the rate in this redshift range in consistent with recent measurements of the star formation rate derived from UV-luminosity densities and IR datasets. Based on 25 type Ia supernovae, we find a SN Ia rate that is a factor 3-5 higher at z~1 compared to earlier estimates at lower redshifts (z<0.5), implying that the type Ia supernova rate traces a higher star formation rate at redshifts z>1 compared to low redshift. At higher redshift (z>1), we find a suggested decrease in the type Ia rate with redshift. This evolution of the Ia rate with redshift is consistent with a type Ia progenitor model where there is a substantial delay between the formation of the progenitor star and the explosion of the supernova. Assuming that the type Ia progenitor stars have initial main sequence masses 3-8 M_Sun, we find that 5-7% of the available progenitors explode as type Ia supernovae.Comment: 16 pages, 3 figures, accepted for publication in the Astrophysical Journa

Solar thermoelectricity Via Advanced Latent Heat Storage

An aspect of the present disclosure is a system that includes a thermal valve having a first position and a second position, a heat transfer fluid, and an energy converter where, when in the first position, the thermal valve prevents the transfer of heat from the heat transfer fluid to the energy converter, and when in the second position, the thermal valve allows the transfer of heat from the heat transfer fluid to the energy converter, such that at least a portion of the heat transferred is converted to electricity by the energy converter

Cooperative regulation of AJM-1 controls junctional integrity in Caenorhabditis elegans epithelia.

The function of epithelial cell sheets depends on the integrity of specialized cell-cell junctions that connect neighbouring cells. We have characterized the novel coiled-coil protein AJM-1, which localizes to an apical junctional domain of Caenorhabditis elegans epithelia basal to the HMR-HMP (cadherin-catenin) complex. In the absence of AJM-1, the integrity of this domain is compromised. Proper AJM-1 localization requires LET-413 and DLG-1, homologues of the Drosophila tumour suppressors Scribble and Discs large, respectively. DLG-1 physically interacts with AJM-1 and is required for its normal apical distribution, and LET-413 mediates the rapid accumulation of both DLG-1 and AJM-1 in the apical domain. In the absence of both dlg-1 and let-413 function AJM-1 is almost completely lost from apical junctions in embryos, whereas HMP-1 (α α-catenin) localization is only mildly affected. We conclude that LET-413 and DLG-1 cooperatively control AJM-1 localization and that AJM-1 controls the integrity of a distinct apical junctional domain in C. elegans. D uring animal development, specialized junctional domains are crucial for the function of epithelial cell sheets. In both vertebrates and invertebrates, adherens junctions are thought to regulate cell-cell adhesion and dynamic changes in cell morphology Here we show that the novel coiled-coil protein AJM-1 (for &apos;apical junction molecule&apos;) is required for the integrity of epithelial junctions of C. elegans and that it localizes to an apical junctional domain. (AJM-1 was originally called JAM-1 (refs 13, 14) but has been renamed to avoid confusion with the vertebrate transmembrane tight junction protein, JAM-1.) This domain is basal to the HMR-HMP(cadherin-catenin) complex; on the basis of the localization of the Discs large homologue DLG-1 to the same domain, it might be required for maintaining a tight apical seal between epithelial cells at apical junctions. Furthermore, we show that AJM-1 directly binds DLG-1, which is required for the proper distribution of AJM-1 around the junctional belt but not for general cell polarity. In addition, we show that in embryos lacking LET-413 the patterns of both DLG-1 and AJM-1 are equally disrupted, including a delay in concentration of these proteins at a narrow apical domain. Almost complete loss of junctional AJM-1 is observed in the absence of both LET-413 and DLG-1, whereas HMP-1 (α-catenin) localization is reduced but junctional. We propose a model in which LET-413 and DLG-1 control the integrity of a distinct apical subdomain by cooperatively regulating the localization of AJM-1. Results AJM-1 encodes a novel coiled-coil protein localizing to C. elegans apical junctions. As an initial step in understanding the molecular composition of apical junctions in C. elegans, we characterized the antigen recognized by the MH27 antibody. The antibody had been previously shown to stain apical borders of C. elegans epitheli

Cooperative regulation of AJM-1 controls junctional integrity in Caenorhabditis elegans epithelia.

The function of epithelial cell sheets depends on the integrity of specialized cell-cell junctions that connect neighbouring cells. We have characterized the novel coiled-coil protein AJM-1, which localizes to an apical junctional domain of Caenorhabditis elegans epithelia basal to the HMR-HMP (cadherin-catenin) complex. In the absence of AJM-1, the integrity of this domain is compromised. Proper AJM-1 localization requires LET-413 and DLG-1, homologues of the Drosophila tumour suppressors Scribble and Discs large, respectively. DLG-1 physically interacts with AJM-1 and is required for its normal apical distribution, and LET-413 mediates the rapid accumulation of both DLG-1 and AJM-1 in the apical domain. In the absence of both dlg-1 and let-413 function AJM-1 is almost completely lost from apical junctions in embryos, whereas HMP-1 (α α-catenin) localization is only mildly affected. We conclude that LET-413 and DLG-1 cooperatively control AJM-1 localization and that AJM-1 controls the integrity of a distinct apical junctional domain in C. elegans. D uring animal development, specialized junctional domains are crucial for the function of epithelial cell sheets. In both vertebrates and invertebrates, adherens junctions are thought to regulate cell-cell adhesion and dynamic changes in cell morphology Here we show that the novel coiled-coil protein AJM-1 (for &apos;apical junction molecule&apos;) is required for the integrity of epithelial junctions of C. elegans and that it localizes to an apical junctional domain. (AJM-1 was originally called JAM-1 (refs 13, 14) but has been renamed to avoid confusion with the vertebrate transmembrane tight junction protein, JAM-1.) This domain is basal to the HMR-HMP(cadherin-catenin) complex; on the basis of the localization of the Discs large homologue DLG-1 to the same domain, it might be required for maintaining a tight apical seal between epithelial cells at apical junctions. Furthermore, we show that AJM-1 directly binds DLG-1, which is required for the proper distribution of AJM-1 around the junctional belt but not for general cell polarity. In addition, we show that in embryos lacking LET-413 the patterns of both DLG-1 and AJM-1 are equally disrupted, including a delay in concentration of these proteins at a narrow apical domain. Almost complete loss of junctional AJM-1 is observed in the absence of both LET-413 and DLG-1, whereas HMP-1 (α-catenin) localization is reduced but junctional. We propose a model in which LET-413 and DLG-1 control the integrity of a distinct apical subdomain by cooperatively regulating the localization of AJM-1. Results AJM-1 encodes a novel coiled-coil protein localizing to C. elegans apical junctions. As an initial step in understanding the molecular composition of apical junctions in C. elegans, we characterized the antigen recognized by the MH27 antibody. The antibody had been previously shown to stain apical borders of C. elegans epitheli

The Carnegie Supernova Project: First Near-Infrared Hubble Diagram to z~0.7

The Carnegie Supernova Project (CSP) is designed to measure the luminosity distance for Type Ia supernovae (SNe Ia) as a function of redshift, and to set observational constraints on the dark energy contribution to the total energy content of the Universe. The CSP differs from other projects to date in its goal of providing an I-band {rest-frame} Hubble diagram. Here we present the first results from near-infrared (NIR) observations obtained using the Magellan Baade telescope for SNe Ia with 0.1 < z < 0.7. We combine these results with those from the low-redshift CSP at z <0.1 (Folatelli et al. 2009). We present light curves and an I-band Hubble diagram for this first sample of 35 SNe Ia and we compare these data to 21 new SNe Ia at low redshift. These data support the conclusion that the expansion of the Universe is accelerating. When combined with independent results from baryon acoustic oscillations (Eisenstein et al. 2005), these data yield Omega_m = 0.27 +/- 0.0 (statistical), and Omega_DE = 0.76 +/- 0.13 (statistical) +/- 0.09 (systematic), for the matter and dark energy densities, respectively. If we parameterize the data in terms of an equation of state, w, assume a flat geometry, and combine with baryon acoustic oscillations, we find that w = -1.05 +/- 0.13 (statistical) +/- 0.09 (systematic). The largest source of systematic uncertainty on w arises from uncertainties in the photometric calibration, signaling the importance of securing more accurate photometric calibrations for future supernova cosmology programs. Finally, we conclude that either the dust affecting the luminosities of SNe Ia has a different extinction law (R_V = 1.8) than that in the Milky Way (where R_V = 3.1), or that there is an additional intrinsic color term with luminosity for SNe Ia independent of the decline rate.Comment: 44 pages, 23 figures, 9 tables; Accepted for publication in the Astrophysical Journa

Fenoldopam use in a burn intensive care unit: a retrospective study

<p>Abstract</p> <p>Background</p> <p>Fenoldopam mesylate is a highly selective dopamine-1 receptor agonist approved for the treatment of hypertensive emergencies that may have a role at low doses in preserving renal function in those at high risk for or with acute kidney injury (AKI). There is no data on low-dose fenoldopam in the burn population. The purpose of our study was to describe our use of low-dose fenoldopam (0.03-0.09 μg/kg/min) infusion in critically ill burn patients with AKI.</p> <p>Methods</p> <p>We performed a retrospective analysis of consecutive patients admitted to our burn intensive care unit (BICU) with severe burns from November 2005 through September 2008 who received low-dose fenoldopam. Data obtained included systolic blood pressure, serum creatinine, vasoactive medication use, urine output, and intravenous fluid. Patients on concomitant continuous renal replacement therapy were excluded. Modified inotrope score and vasopressor dependency index were calculated. One-way analysis of variance with repeated measures, Wilcoxson signed rank, and chi-square tests were used. Differences were deemed significant at p < 0.05.</p> <p>Results</p> <p>Seventy-seven patients were treated with low-dose fenoldopam out of 758 BICU admissions (10%). Twenty (26%) were AKI network (AKIN) stage 1, 14 (18%) were AKIN stage 2, 42 (55%) were AKIN stage 3, and 1 (1%) was AKIN stage 0. Serum creatinine improved over the first 24 hours and continued to improve through 48 hours (<it>p </it>< 0.05). There was an increase in systolic blood pressure in the first 24 hours that was sustained through 48 hours after initiation of fenoldopam (<it>p </it>< 0.05). Urine output increased after initiation of fenoldopam without an increase in intravenous fluid requirement (<it>p </it>< 0.05; <it>p </it>= NS). Modified inotrope score and vasopressor dependency index both decreased over 48 hours (<it>p </it>< 0.0001; <it>p </it>= 0.0012).</p> <p>Conclusions</p> <p>These findings suggest that renal function was preserved and that urine output improved without a decrease in systolic blood pressure, increase in vasoactive medication use, or an increase in resuscitation requirement in patients treated with low-dose fenoldopam. A randomized controlled trial is required to establish the efficacy of low-dose fenoldopam in critically ill burn patients with AKI.</p