PubMed Health⌕ Search

Biomedical subjects

R Iyengar

Publications and source records attributed to R Iyengar.

At least 19 recordsLinked to original sources
PubMed2003

Purification of human natural killer cells using a clinical-scale immunomagnetic method.

BACKGROUND: Infection, graft failure, disease relapse, and GvHD are significant adverse events associated with allogeneic BMT. Although donor leukocyte infusion has been used to prevent or to treat infection, graft failure, and relapse, the potential clinical benefits are often outweighed by the risk of T cell-mediated GvHD. Results from animal studies suggest that donor natural killer (NK) cells may be an ideal cell type for prevention or treatment of these adverse events. We have therefore sought to develop an automated, efficient, and clinical-scale human NK cell-purification method. METHODS: Twelve leukopheresis products were purified for NK cells using a two-step immunomagnetic method. CD3(+) cells were first depleted from the apheresis products. CD56(+) cells were then enriched from the CD3(+) cell-depleted products. RESULTS: The median percentage of CD3(-)CD56(+) NK cells in the final products was 91.0%, and the median recovery was 48.7%. The median depletion for CD3(+)CD56(-) T cells was 5.3 log. Natural cytotoxicity of the purified cells was approximately five-fold higher than that of unpurified mononuclear cells, and it could be further increased by stimulation of the purified cell with IL2. DISCUSSION: We described a large-scale purification method for automated, efficient, and rapid isolation of human NK cells that yielded minimal contamination with T cells or B cells. These purified NK cells may be expedient for preclinical and clinical uses.

Antigens, CD19↗
PubMed2001

The signal transfer regions of G alpha(s).

The crystal structure of soluble functional fragments of adenylyl cyclase complexed with G alpha(s) and forskolin, shows three regions of G alpha(s) in direct contact with adenylyl cyclase. The functions of these three regions are not known. We tested synthetic peptides encoding these regions of G alpha(s) on the activities of full-length adenylyl cyclases 2 and 6. A peptide encoding the Switch II region (amino acids 222-247) stimulated both adenylyl cyclases 2- to 3-fold. Forskolin synergized the stimulation. Addition of peptides in the presence of activated G alpha(s) partially inhibited G alpha(s) stimulation. Corresponding Switch II region peptides from G alpha(q) and G alpha(i) did not stimulate adenylyl cyclase. A peptide encoding the Switch I region (amino acids 199-216) also stimulated AC2 and AC6. The stimulatory effects of the two peptides at saturating concentrations were non-additive. A peptide encoding the third contact region (amino acids 268-286) located in the alpha 3-beta 5 region, inhibits basal, forskolin, and G alpha(s)-stimulated enzymatic activities. Since this region in G alpha(s) interacts with both the central cytoplasmic loop and C-terminal tail of adenylyl cyclases this peptide may be involved in blocking interactions between these two domains. These functional data in conjunction with the available structural information suggest that G alpha(s) activation of adenylyl cyclase is a complex event where the alpha 3-beta 5 loop of G alpha(s) may bring together the central cytoplasmic loop and C-terminal tail of adenylyl cyclase thus allowing the Switch I and Switch II regions to function as signal transfer regions to activate adenylyl cyclase.

Adenylyl Cyclases↗
PubMed2001

Mitogen-activated protein kinase regulates early phosphorylation and delayed expression of Ca2+/calmodulin-dependent protein kinase II in long-term potentiation.

Activation of mitogen-activated protein kinase (MAPK) and Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) are required for numerous forms of neuronal plasticity, including long-term potentiation (LTP). We induced LTP in rat hippocampal area CA1 using theta-pulse stimulation (TPS) paired with beta-adrenergic receptor activation [isoproterenol (ISO)], a protocol that may be particularly relevant to normal patterns of hippocampal activity during learning. This stimulation resulted in a transient phosphorylation of p42 MAPK, and the resulting LTP was MAPK dependent. In addition, CaMKII was regulated in two, temporally distinct ways after TPS-ISO: a transient rise in the fraction of phosphorylated CaMKII and a subsequent persistent increase in CaMKII expression. The increases in MAPK and CaMKII phosphorylation were strongly colocalized in the dendrites and cell bodies of CA1 pyramidal cells, and both the transient phosphorylation and delayed expression of CaMKII were prevented by inhibiting p42/p44 MAPK. These results establish a novel bimodal regulation of CaMKII by MAPK, which may contribute to both post-translational modification and increased gene expression.

Adrenergic beta-Agonists↗
PubMed2001

Modular design of Gbeta as the basis for reversible specificity in effector stimulation.

The G protein Gbetagamma subunit complex stimulates effectors by direct interactions utilizing extensive Gbeta regions over the surface of its propeller structure that faces the Galpha subunit. Our previous experiments have shown the resolved functions of signal transfer and general binding for Gbeta regions involved in stimulation of the effector phospholipase C-beta2, PLC-beta2, within the region Gbeta-(86-135), which comprises three beta strands arranged in a structurally contiguous fashion (Buck, E., Li, J., Chen, Y., Weng, G., Sacarlata, S., and Iyengar, R. (1999) Science 283, 1332-1335). This raises an important question as to why mutagenesis studies indicate that an extensive set of sites all over the Gbeta propeller structure and outside the 86-135 region are involved in Gbeta regulation of PLC-beta2. Using peptides to define functions of these Gbeta regions, we find that Gbeta signaling to PLC-beta2 relies on a collection of modular signal transfer and general binding units, each with lower apparent affinity relative to Gbetagamma-PLC interactions. Gbeta-(42-54) functions as a signal transfer region, Gbeta-(228-249) and Gbeta-(321-340) function in general binding, and Gbeta-(64-84) and Gbeta-(300-313) seem to play a structural role rather than a direct contact with the effector. A substitution within the Gbeta-(42-54) signal transfer region that increases the K(act) of this peptide for PLC-beta2 is accompanied by an increase in the observed maximal extent of signal transfer. We conclude that the lower K(act) for individual signal transfer regions may result in a decrease in the maximal effect of signal transfer. The spatial resolution of the signal transfer and general binding regions over a wide surface of Gbeta allow geometrical constraints to achieve specificity even with relatively low affinity interactions.

GTP-Binding Proteins↗
PubMed2001

G protein coupled receptor signaling through the Src and Stat3 pathway: role in proliferation and transformation.

Extracellular signals when routed through signaling pathways that use heterotrimeric G proteins can engage multiple signaling pathways leading to diverse biological consequences. One locus at which signal sorting occurs is at the level of G proteins. G protein alpha-subunits appear to be capable of interacting with different effectors leading to engagement of distinct signaling pathways. Regulation of different pathways in turn leads to different biological outcomes. The process of neoplastic transformation is controlled to a large extent through the activation and inhibition of signaling pathways. Signaling pathways such as the Ras-MAPK, v-Src-Stat3 pathways are activated in the process of transformation. Expression of activated Galpha subunits have been shown to cause transformation of cells. While activation of the MAPK 1,2 pathway by various Galpha subunits has been reported for several years, recent studies show the activation and involvement of Src and Stat3 pathways in Galphao and Galphai mediated transformation of cells. Recent studies also suggest that both Galphai and Galphas may be able to interact with and activate Src. The activation of Src and Stat3 by G proteins has also been demonstrated by ligand-induced activation of G protein receptors. So increasingly it is becoming clear that the Src and Stat3 pathways are potential effectors for G proteins and that they may play a role in G protein function.

Cell Division↗
PubMed2001

Expression of Q227L-Galpha(s) inhibits intimal vessel wall hyperplasia after balloon injury.

Interaction between signaling pathways regulates many cellular functions, including proliferation. The Galpha(s)/cAMP pathway is known to inhibit signal flow from receptor tyrosine kinases to mitogen-activated protein kinase (MAPK)-1,2 and, thus, inhibit proliferation. Elevation of cAMP or adenovirus-directed expression of mutant (Q227L)-Galpha(s) (alpha(s)*) inhibits the proliferation of rat vascular smooth muscle cells (VSMCs) in culture. Platelet-derived growth factor (PDGF) stimulated MAPK activation and DNA synthesis was also blocked by expression of alpha(s)*. However, it is not known whether such mechanisms are operative in vivo. Proliferation of vascular smooth muscle cells in vivo was induced by balloon injury of carotid arteries in the rat. Recombinant adenovirus encoding beta-galactosidase (beta-gal) or alpha(s)* was applied to arterial segments injured by the balloon catheters. The alpha(s)*-treated vessels showed decreased phospho-MAPK staining in the intima as compared with beta-gal-treated vessels. Application of alpha(s)*, but not beta-gal containing adenovirus, inhibited formation of neointima by 50%. No change was observed in total vessel diameter or in the media or adventitia. These results suggest that the interaction between the Galpha(s) and MAPK pathways can regulate proliferation in vivo and that targeted expression of activated Galpha(s) may have therapeutic potential for the treatment of vascular pathophysiologies that arise from intimal hyperplasia.

Adenoviridae↗
PubMed2001

Functional modules in biological signalling networks.

Signalling pathways carry information from the outside of the cell to cellular machinery capable of producing biochemical or physiological responses. Although linear signalling plays an important role in biological regulation, signalling pathways are often interconnected to form networks. We have used computational analysis to study emergent properties of simple networks that consist of up to four pathways, We find that when one pathway gates signal flow through other pathways which produce physiological responses, gating results in signal prolongation such that the signal may be consolidated into a physiological response. When two pathways combine to form a feedback loop such feedback loops can exhibit bistability. Negative regulators of the loop can serve as the locus for flexibility whereby the system has the capability of switching states or functioning as a proportional read-out system. Networks where bistable feedback loops are connected to gates can lead to persistent signal activation at distal locations. These emergent properties indicate system analysis of signalling networks may be useful in understanding higher-order biological functions.

Animals↗
PubMed2000

Long-term potentiation induced by theta frequency stimulation is regulated by a protein phosphatase-1-operated gate.

Long-term potentiation (LTP) can be induced in the Schaffer collateral-->CA1 synapse of hippocampus by stimulation in the theta frequency range (5-12 Hz), an effect that depends on activation of the cAMP pathway. We investigated the mechanisms of the cAMP contribution to this form of LTP in the rat hippocampal slice preparation. theta pulse stimulation (TPS; 150 stimuli at 10 Hz) by itself did not induce LTP, but the addition of either the beta-adrenergic agonist isoproterenol or the cAMP analog 8-bromo-cAMP (8-Br-cAMP) enabled TPS-induced LTP. The isoproterenol effect was blocked by postsynaptic inhibition of cAMP-dependent protein kinase. Several lines of evidence indicated that cAMP enabled LTP by blocking postsynaptic protein phosphatase-1 (PP1). Activators of the cAMP pathway reduced PP1 activity in the CA1 region and increased the active form of inhibitor-1, an endogenous inhibitor of PP1. Postsynaptic injection of activated inhibitor-1 mimicked the LTP-enabling effect of cAMP pathway stimulation. TPS evoked complex spiking when isoproterenol was present. However, complex spiking was not sufficient to enable TPS-induced LTP, which additionally required the inhibition of postsynaptic PP1. PP1 inhibition seems to promote the activation of Ca(2+)/calmodulin-dependent protein kinase (CaMKII), because (1) a CaMKII inhibitor blocked the induction of LTP by TPS paired with either isoproterenol or activated inhibitor-1 and (2) CaMKII in area CA1 was activated by the combination of TPS and 8-Br-cAMP but not by either stimulus alone. These results indicate that the cAMP pathway enables TPS-induced LTP by inhibiting PP1, thereby enhancing Ca(2+)-independent CaMKII activity.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed2000

Synthesis and use of 3'-(azidoiodosalicyl) derivatives of 2', 5'-dideoxyadenosine as photoaffinity ligands for adenylyl cyclase.

3'-[(4-Azidosalicyl)glycyl]-2',5'-dideoxyadenosine (1), 3'- [(4-azidosalicyl)-gamma-aminobutyryl]-2',5'-dideoxyadenosine (2), and the (125)I-labeled mono- and diiodinated analogs of 1 were synthesized and tested as photoaffinity probes for adenylyl cyclases. Kinetics for inhibition of purified type I enzyme by 1 was noncompetitive with respect to Mn(*)5'-ATP in the absence of light, implying a P-site mechanism of inhibition. In a UV-dependent manner both 1 and 2 and the iodinated derivative of 1 irreversibly inactivated membrane-bound and purified forms of recombinant type I bovine adenylyl cyclase expressed in ovarian cells of either the fall armyworm (Sf9) or Trichoplasia ni (High Five). Irreversible inactivation was independent of 5'-ATP and was prevented by 2', 5'-dideoxyadenosine. Adenylyl cyclase, whether purified from bovine brain or in membranes from High Five cells expressing type I enzyme, when subjected to UV irradiation in the presence of (125)I-labeled 1 resulted in radioactive incorporation into protein migrating at approximately 116 kDa. The cross-linking of 1 and its iodinated derivative with adenylyl cyclase suggests potential for such compounds to be useful in structural studies of adenylyl cyclases or of other proteins for which adenine nucleosides are substrates or allosteric regulators.

Adenosine↗
PubMed2000

Stat3-mediated transformation of NIH-3T3 cells by the constitutively active Q205L Galphao protein.

Expression of Q205L Galphao (Galphao*), an alpha subunit of heterotrimeric guanine nucleotide-binding proteins (G proteins) that lacks guanosine triphosphatase (GTPase) activity in NIH-3T3 cells, results in transformation. Expression of Galphao* in NIH-3T3 cells activated signal transducer and activator of transcription 3 (Stat3) but not mitogen-activated protein (MAP) kinases 1 or 2. Coexpression of dominant negative Stat3 inhibited Galphao*-induced transformation of NIH-3T3 cells and activation of endogenous Stat3. Furthermore, Galphao* expression increased activity of the tyrosine kinase c-Src, and the Galphao*-induced activation of Stat3 was blocked by expression of Csk (carboxyl-terminal Src kinase), which inactivates c-Src. The results indicate that Stat3 can function as a downstream effector for Galphao* and mediate its biological effects.

3T3 Cells↗
PubMed1999

Modulation of rap activity by direct interaction of Galpha(o) with Rap1 GTPase-activating protein.

We used the yeast two-hybrid system to identify proteins that interact directly with Galpha(o). Mutant-activated Galpha(o) was used as the bait to screen a cDNA library from chick dorsal root ganglion neurons. We found that Galpha(o) interacted with several proteins including Gz-GTPase-activating protein (Gz-GAP), a new RGS protein (RGS-17), a novel protein of unknown function (IP6), and Rap1GAP. This study focuses on Rap1GAP, which selectively interacts with Galpha(o) and Galpha(i) but not with Galpha(s) or Galpha(q). Rap1GAP interacts more avidly with the unactivated Galpha(o) as compared with the mutant (Q205L)-activated Galpha(o). When expressed in HEK-293 cells, unactivated Galpha(o) co-immunoprecipitates with the Rap1GAP. Expression of chick Rap1GAP in PC-12 cells inhibited activation of Rap1 by forskolin. When unactivated Galpha(o) was expressed, the amount of activated Rap1 was greatly increased. This effect was not observed with the Q205L-Galpha(o). Expression of unactivated Galpha(o) stimulated MAP-kinase (MAPK1/2) activity in a Rap1GAP-dependent manner. These results identify a novel function of Galpha(o), which in its resting state can sequester Rap1GAP thereby regulating Rap1 activity and consequently gating signal flow from Rap1 to MAPK1/2. Thus, activation of G(o) could modulate the Rap1 effects on a variety of cellular functions.

Amino Acid Substitution↗
PubMed1999

Complexity in biological signaling systems.

Biological signaling pathways interact with one another to form complex networks. Complexity arises from the large number of components, many with isoforms that have partially overlapping functions; from the connections among components; and from the spatial relationship between components. The origins of the complex behavior of signaling networks and analytical approaches to deal with the emergent complexity are discussed here.

Animals↗
PubMed1999

Resolution of a signal transfer region from a general binding domain in gbeta for stimulation of phospholipase C-beta2.

Signaling by guanine nucleotide-binding proteins (G proteins) involves sequential protein-protein interactions. G protein-betagamma subunit (Gbetagamma) interactions with phospholipase C-beta2 (PLC-beta2) were studied to determine if all Gbeta contacts are required for signaling. A peptide encoding Gbeta amino acid residues 86 to 105 stimulated PLC-beta2. Six residues (96 to 101) within this sequence could transfer signals and thus constitute a core signal transfer region. Another peptide, encoding Gbeta amino acid residues 115 to 135, did not substantially stimulate PLC-beta2 by itself but inhibited Gbetagamma stimulation, indicating that residues 115 to 135 constitute a general binding domain. Resolution of signal transfer regions from general binding domains indicates that all protein-protein contacts are not required for signal transfer and that it may be feasible to synthesize agonists and antagonists that regulate intracellular signal flow.

Amino Acid Substitution↗
PubMed1999

Emergent properties of networks of biological signaling pathways.

Many distinct signaling pathways allow the cell to receive, process, and respond to information. Often, components of different pathways interact, resulting in signaling networks. Biochemical signaling networks were constructed with experimentally obtained constants and analyzed by computational methods to understand their role in complex biological processes. These networks exhibit emergent properties such as integration of signals across multiple time scales, generation of distinct outputs depending on input strength and duration, and self-sustaining feedback loops. Feedback can result in bistable behavior with discrete steady-state activities, well-defined input thresholds for transition between states and prolonged signal output, and signal modulation in response to transient stimuli. These properties of signaling networks raise the possibility that information for "learned behavior" of biological systems may be stored within intracellular biochemical reactions that comprise signaling pathways.

Animals↗
PubMed1998

Identity of adenylyl cyclase isoform determines the rate of cell cycle progression in NIH 3T3 cells.

Cell cycle progression is regulated by cAMP in several cell types. Cellular cAMP levels depend on the activity of different adenylyl cyclases (ACs), which have varied signal-receiving capabilities. The role of individual ACs in regulating proliferative responses was investigated. Native NIH 3T3 cells contain AC6, an isoform that is inhibited by a variety of signals. Proliferation of exogenous AC6-expressing cells was the same as in control cells. In contrast, expression of AC2, an isoform stimulated by protein kinase C (PKC), resulted in inhibition of cell cycle progression and increased doubling time. In AC2-expressing cells, platelet-derived growth factor (PDGF) elevated cAMP levels in a PKC-dependent manner. PDGF stimulation of mitogen-activated protein kinases 1 and 2 (MAPK 1,2), DNA synthesis, and cyclin D1 expression was reduced in AC2-expressing cells as compared with control cells. Dominant negative protein kinase A relieved the AC2 inhibition of PDGF-induced DNA synthesis. Expression of AC2 also blocked H-ras-induced transformation of NIH 3T3 cells. These observations indicate that, because AC2 is stimulated by PKC, it can be activated by PDGF concurrently with the stimulation of MAPK 1,2. The elevation in cAMP results in inhibition of signal flow from the PDGF receptor to MAPK 1,2 and a significant reduction in the proliferative response to PDGF. Thus, the molecular identity and signal receiving capability of the AC isoforms in a cell could be important for proliferative homeostasis.

3T3 Cells↗
PubMed1998

Gating of CaMKII by cAMP-regulated protein phosphatase activity during LTP.

Long-term potentiation (LTP) at the Schaffer collateral-CA1 synapse involves interacting signaling components, including calcium (Ca2+)/calmodulin-dependent protein kinase II (CaMKII) and cyclic adenosine monophosphate (cAMP) pathways. Postsynaptic injection of thiophosphorylated inhibitor-1 protein, a specific inhibitor of protein phosphatase-1 (PP1), substituted for cAMP pathway activation in LTP. Stimulation that induced LTP triggered cAMP-dependent phosphorylation of endogenous inhibitor-1 and a decrease in PP1 activity. This stimulation also increased phosphorylation of CaMKII at Thr286 and Ca2+-independent CaMKII activity in a cAMP-dependent manner. The blockade of LTP by a CaMKII inhibitor was not overcome by thiophosphorylated inhibitor-1. Thus, the cAMP pathway uses PP1 to gate CaMKII signaling in LTP.

Animals↗