Paramjeet Randhawa

Publications

• 9.21

  Impact points

  The Ras activator RasGRP3 mediates diabetes-induced embryonic defects and affects endothelial cell migration.

  Paramjeet K Randhawa, Svetlana Rylova, Jessica Y Heinz, Stephanie Kiser, Joanna H Fried, William P Dunworth, Amanda L Anderson, Andrew T Barber, John C Chappell, David M Roberts, Victoria L Bautch

  Circulation research. 05/2011; 108(10):1199-208.

  Fetuses that develop in diabetic mothers have a higher incidence of birth defects that include cardiovascular defects, but the signaling pathways that mediate these developmental effects are poorly understood. It is reasonable to hypothesize that diabetic maternal effects are mediated by 1 or more p... [more] Fetuses that develop in diabetic mothers have a higher incidence of birth defects that include cardiovascular defects, but the signaling pathways that mediate these developmental effects are poorly understood. It is reasonable to hypothesize that diabetic maternal effects are mediated by 1 or more pathways activated downstream of aberrant glucose metabolism, because poorly controlled maternal glucose levels correlate with the frequency and severity of the defects. We investigated whether RasGRP3 (Ras guanyl-releasing protein 3), a Ras activator expressed in developing blood vessels, mediates diabetes-induced vascular developmental defects. RasGRP3 is activated by diacylglycerol, and diacylglycerol is overproduced by aberrant glucose metabolism in diabetic individuals. We also investigated the effects of overactivation and loss of function for RasGRP3 in primary endothelial cells and developing vessels. Analysis of mouse embryos from diabetic mothers showed that diabetes-induced developmental defects were dramatically attenuated in embryos that lacked Rasgrp3 function. Endothelial cells that expressed activated RasGRP3 had elevated Ras-ERK signaling and perturbed migration, whereas endothelial cells that lacked Rasgrp3 function had attenuated Ras-ERK signaling and did not migrate in response to endothelin-1. Developing blood vessels exhibited endothelin-stimulated vessel dysmorphogenesis that required Rasgrp3 function. These findings provide the first evidence that RasGRP3 contributes to developmental defects found in embryos that develop in a diabetic environment. The results also elucidate RasGRP3-mediated signaling in endothelial cells and identify endothelin-1 as an upstream input and Ras/MEK/ERK as a downstream effector pathway. RasGRP3 may be a novel therapeutic target for the fetal complications of diabetes.
• 1.90

  Impact points

  Chapter 6 in vitro differentiation of mouse embryonic stem cells into primitive blood vessels.

  Svetlana N Rylova, Paramjeet K Randhawa, Victoria L Bautch

  Methods in enzymology. 02/2008; 443:103-17.

  Mouse embryonic stem (ES) cells, derived from the inner cell mass of blastocyst stage embryos, undergo programmed differentiation in vitro to form a primitive vasculature. This programmed differentiation proceeds through similar processes of vasculogenesis and angiogenesis found during early vascula... [more] Mouse embryonic stem (ES) cells, derived from the inner cell mass of blastocyst stage embryos, undergo programmed differentiation in vitro to form a primitive vasculature. This programmed differentiation proceeds through similar processes of vasculogenesis and angiogenesis found during early vascular development in vivo. Partially differentiated ES cell clumps or embryoid bodies (EBs) first form blood islands that are subsequently transformed into a network of primitive blood vessels that contain lumens. Therefore, vascular differentiation of ES cells is an ideal model to study and manipulate early vascular development. Here we provide protocols for the routine maintenance of mouse ES cells and in vitro differentiation. We also include protocols for establishing transgenic ES cell lines and visualization of blood vessels by use of endothelial specific molecular markers.
• 2.55

  Impact points

  Peptide rescues GLUT4 recruitment, but not GLUT4 activation, in insulin resistance.

  Makoto Funaki, Kate Benincasa, Paramjeet K Randhawa

  Biochemical and biophysical research communications. 10/2007; 360(4):891-6.

  Insulin-stimulated GLUT4 recruitment to the plasma membrane is impaired in insulin resistance. We recently reported that a cell permeable phosphoinositide-binding peptide induces GLUT4 recruitment as potently as insulin, but does not activate GLUT4 to initiate glucose uptake. Here we investigated wh... [more] Insulin-stimulated GLUT4 recruitment to the plasma membrane is impaired in insulin resistance. We recently reported that a cell permeable phosphoinositide-binding peptide induces GLUT4 recruitment as potently as insulin, but does not activate GLUT4 to initiate glucose uptake. Here we investigated whether the peptide-induced GLUT4 recruitment is intact in insulin resistance. The expression levels of GLUT1 and GLUT4 were unaffected by chronically treating 3T3-L1 adipocytes with insulin. GLUT4 recruitment by acute insulin stimulation after chronic insulin treatment was significantly reduced, but was fully restored by the peptide treatment. However, subsequent acute insulin stimulation to activate GLUT4 failed to increase glucose uptake in peptide-pretreated cells. Insulin-stimulated GLUT1 recruitment was unaffected by the peptide pretreatment. These results suggest that the GLUT4 recruitment signal caused by the peptide is intact in insulin resistance, but GLUT4 activation that occurs subsequent to recruitment is not rescued by the peptide treatment.
• 6.06

  Impact points

  Separation of insulin signaling into distinct GLUT4 translocation and activation steps.

  Makoto Funaki, Paramjeet Randhawa, Paul A Janmey

  Molecular and cellular biology. 10/2004; 24(17):7567-77.

  GLUT4 (glucose transporter 4) plays a pivotal role in insulin-induced glucose uptake to maintain normal blood glucose levels. Here, we report that a cell-permeable phosphoinositide-binding peptide induced GLUT4 translocation to the plasma membrane without inhibiting IRAP (insulin-responsive aminopep... [more] GLUT4 (glucose transporter 4) plays a pivotal role in insulin-induced glucose uptake to maintain normal blood glucose levels. Here, we report that a cell-permeable phosphoinositide-binding peptide induced GLUT4 translocation to the plasma membrane without inhibiting IRAP (insulin-responsive aminopeptidase) endocytosis. However, unlike insulin treatment, the peptide treatment did not increase glucose uptake in 3T3-L1 adipocytes, indicating that GLUT4 translocation and activation are separate events. GLUT4 activation can occur at the plasma membrane, since insulin was able to increase glucose uptake with a shorter time lag when inactive GLUT4 was first translocated to the plasma membrane by pretreating the cells with this peptide. Inhibition of phosphatidylinositol (PI) 3-kinase activity failed to inhibit GLUT4 translocation by the peptide but did inhibit glucose uptake when insulin was added following peptide treatment. Insulin, but not the peptide, stimulated GLUT1 translocation. Surprisingly, the peptide pretreatment inhibited insulin-induced GLUT1 translocation, suggesting that the peptide treatment has both a stimulatory effect on GLUT4 translocation and an inhibitory effect on insulin-induced GLUT1 translocation. These results suggest that GLUT4 requires translocation to the plasma membrane, as well as activation at the plasma membrane, to initiate glucose uptake, and both of these steps normally require PI 3-kinase activation.
• 4.80

  Impact points

  Antibacterial activities of rhodamine B-conjugated gelsolin-derived peptides compared to those of the antimicrobial peptides cathelicidin LL37, magainin II, and melittin.

  Robert Bucki, Jennifer J Pastore, Paramjeet Randhawa, Rolands Vegners, Daniel J Weiner, Paul A Janmey

  Antimicrobial agents and chemotherapy. 06/2004; 48(5):1526-33.

  The growing number of antibiotic-resistant bacteria necessitates the search for new antimicrobial agents and the principles by which they work. We report that cell membrane-permeant rhodamine B (RhB)-conjugated peptides based on the phosphatidylinositol-4,5-bisphosphate binding site of gelsolin can ... [more] The growing number of antibiotic-resistant bacteria necessitates the search for new antimicrobial agents and the principles by which they work. We report that cell membrane-permeant rhodamine B (RhB)-conjugated peptides based on the phosphatidylinositol-4,5-bisphosphate binding site of gelsolin can kill the gram-negative organisms Escherichia coli and Pseudomonas aeruginosa and the gram-positive organism Streptococcus pneumoniae. RhB linkage to the QRLFQVKGRR sequence in gelsolin was essential for the antibacterial function, since the unconjugated peptide had no effect on the bacteria tested. Because RhB-QRLFQVKGRR (also termed PBP10), its scrambled sequence (RhB-FRVKLKQGQR), and PBP10 synthesized from D-isomer amino acids show similar antibacterial properties, the physical and chemical properties of these derivatives appear to be more important than specific peptide folding for their antibacterial functions. The similar activities of PBP10 and all-D-amino-acid PBP10 also indicate that a specific interaction between RhB derivatives and bacterial proteins is unlikely to be involved in the bacterial killing function of PBP10. By using a phospholipid monolayer system, we found a positive correlation between the antibacterial function of PBP10, as well as some naturally occurring antibacterial peptides, and the intrinsic surface pressure activity at the hydrophobic-hydrophilic interface. Surprisingly, we observed little or no dependence of the insertion of these peptides into lipid monolayers on the phospholipid composition. These studies show that an effective antimicrobial agent can be produced from a peptide sequence with specificity to a phospholipid not found in bacteria, and comparisons with other antimicrobial agents suggest that the surface activities of these peptides are more important than specific binding to bacterial proteins or lipids for their antimicrobial functions.
• 2.41

  Impact points

  Purification of salmon thrombin and its potential as an alternative to mammalian thrombins in fibrin sealants.

  Sarah E Michaud, Louise Z Wang, Neha Korde, Robert Bucki, Paramjeet K Randhawa, Jennifer J Pastore, Hervé Falet, Karin Hoffmeister, Reet Kuuse, Raivo Uibo, Julia Herod, Evelyn Sawyer, Paul A Janmey

  Thrombosis research. 10/2002; 107(5):245-54.

  A method to produce highly purified thrombin from salmon blood is described, and a series of biochemical, cell biologic, and biophysical assays demonstrate the functional similarities and some differences between salmon and human thrombins. Salmon thrombin with specific activity greater than 1000 un... [more] A method to produce highly purified thrombin from salmon blood is described, and a series of biochemical, cell biologic, and biophysical assays demonstrate the functional similarities and some differences between salmon and human thrombins. Salmon thrombin with specific activity greater than 1000 units/mg total protein can be prepared by modifications of the methods used for purification of human thrombin. Using a synthetic substrate based on the human fibrinogen A-alpha polypeptide sequence as an indicator of enzymatic activity, salmon and human thrombin preparations contain similar specific activities per mass of purified protein. Salmon thrombin activates human fibrinogen and initiates the formation of fibrin clots whose structure and rheologic properties are indistinguishable from those of human fibrin clotted by human thrombin. Salmon thrombin also activates human platelets. Approximately 10 times higher activities are needed for the same rate of platelet aggregation compared to human thrombin, and some aspects of platelet activation, most notably phosphatidylserine exposure, are diminished relative to the effects of human thrombin. This latter finding suggests that salmon thrombin may not activate all of the receptors that are targets of human thrombin, although it does appear to activate signals that are sufficient to produce normal rates of activation and aggregation as measured by conventional aggregometry. Together with the recent purification of salmon fibrinogen and its application in mammalian wound healing, the availability of salmon thrombin allows the formulation of biological sealants devoid of any exogenous mammalian proteins and so may aid the design of materials with increased safety from infectious disease transmission.