Showing items related by title, author, creator and subject.

• Structural and electrochemical properties of wild-type and mutant cytochromes b-(5)

  Sarma, Siddhartha P.; Guiles, Ronald (1996)

  The main objective of this thesis project was to gain a better understanding of the protein structural factors that modulate heme reduction potentials in b-type cytochromes. Rat liver microsomal cytochrome {dollar}b\sb5{dollar} was chosen as a model system because the availability of a synthetic gene coding for the protein permits mutagenic experiments to be performed. The synthetic gene encoding for cytochrome {dollar}b\sb5{dollar} was subcloned into a high yield bacterial overexpression system that enabled isotopic enrichment of proteins. Site directed mutants of cytochrome {dollar}b\sb5{dollar} were prepared by subcloning the synthetic gene into bacteriophage DNA m 13mp18. Mutants of cytochrome {dollar}b\sb5{dollar} were designed to achieve reorientation of individual axial imidazole ligands. The orientation of the axial ligand planes is thought to modulate the reduction potential of bis(imidazole) axially ligated heme proteins. The A67V (alanine to valine) mutation resulted in a reorientation of the H63 imidazole ring and a shift in reduction potential by {dollar}-20{dollar} mV. Structural characterization of the A67V mutant protein was achieved using homonuclear and heteronuclear NMR methods. Calculation of the orientation of the components of paramagnetic susceptibility tensor showed that the wild type and mutant proteins differed only in the orientation of the z-component. The rotation of the z-component of the susceptibility tensor is in the same direction as the rotation of the H63 imidazole ring. EPR and Near-IR data suggest that the stability of iron d-orbital energy levels in the reduced proteins may be significant in determining the reduction potential of b-type heme proteins. Multidimensional double and triple resonance NMR methods have been applied to assign the backbone and side-chain {dollar}\sp{lcub}13{rcub}{dollar}C resonances for both equilibrium conformers of ferricytochrome {dollar}b\sb5{dollar} On the basis observed NOEs and backbone {dollar}\sp{lcub}13{rcub}{dollar}C chemical shifts, the solution secondary structure of cytochrome {dollar}b\sb5{dollar} has been determined. The {dollar}\sp{lcub}13{rcub}{dollar}C chemical shifts of backbone and side-chain atoms are relatively insensitive to paramagnetic effects. The reliability of such methods in anisotropic paramagnetic systems, where large pseudocontact shifts are observed, is evaluated through calculations of the magnitude of such shifts.
• The involvement of RVM ionotropic glutamate receptors in descending modulation of persistent inflammatory hyperalgesia

  Guan, Yun; Dubner, Ronald (2002)

  Descending pathways from the rostral ventromedial medulla (RVM) modulate spinal nociceptive transmission. Descending pain modulation exhibits dynamic functional changes in response to persistent pain. Glutamate receptors in the RVM are involved in descending pain modulation. However, their roles in descending modulation of persistent inflammatory hyperalgesia are unclear. Behavioral studies showed that intra-RVM microinjection of NMDA produced descending modulation that depended upon the post-inflammatory time period. At 3 hr post-inflammation, NMDA induced facilitation at a lower dose (10 pmol) and an inhibition at a higher dose (1000 pmol). At 24 hr post-inflammation, NMDA (0.1--1000 pmol) produced a dose-dependent inhibition. AMPA (0.1--100 pmol) produced only dose-dependent inhibition at both 3 hr and 24 hr. Importantly, there is a leftward shift in dose-response curves of NMDA- and AMPA-produced modulation at 24 hr post-inflammation, compared to that at 3 hr, suggesting a time-dependent increase in glutamatergic neurotransmission in the RVM after inflammation.;We further identified the molecular mechanisms underlying the enhanced AMPA receptor function. RT-PCR analysis indicated an upregulation of GluR1-flip (5 hr--24 hr), GluR2-flip (24 hr) and GluR2-flop (24 hr) mRNAs in the RVM after inflammation. Western blots demonstrated that GluR1 protein levels were upregulated at 24 hr-3 days post-inflammation. GluR2 protein levels remained unchanged after inflammation. Immunohistochemistry demonstrated an increase in GluR1-like immunoreactivity localized to the RVM at 24 hr post-inflammation. Phosphorylation of GluR1 Ser831 plays an important role in regulation of AMPA receptor function. Western blots indicated a rapid and prolonged increase in phospho-Ser831 GluR1 levels in the RVM after inflammation. The onset of upregulation of GluR1 phosphorylation was blocked by pre-emptive local anesthesia of the injured area and attenuated by pretreatment of NMDA receptor antagonists, PKC and CaMKII inhibitors in the RVM. These findings suggest that AMPA receptors in the RVM undergo selective transcriptional, translational and post-translational modulations following inflammation. These findings support the notion that there is a time-dependent dynamic change in descending modulation after inflammation. This study demonstrates that ionotropic glutamate receptors in the RVM are involved in the descending modulation of inflammatory hyperalgesia and play a role in plasticity in descending pain modulation after persistent pain.
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  Regulation of translation initiation upon B-cell receptor activation

  Steinhardt, James John; Gartenhaus, Ronald B. (2014)

  Eukaryotic initiation factor 4A (eIF4A) promotes the translation initiation of genes harboring 5' untranslated regions (UTRs) with complex secondary structures. eIF4A activity is modulated by its inhibitor, programmed cell death 4 (PDCD4), which is regulated by the AKT pathway in B-cells. The release and degradation of PDCD4 frees eIF4A to bind to eIF4G, which together with the m7GTP cap-binding protein, eIF4E, forms the eIF4F cap-binding complex. Human diffuse large B-cell lymphomas (DLBCLs) are derived from antigen experienced B-cells and often display high expression of oncogenes that contain complex secondary structures in their 5' UTRs. We hypothesized that activated B-cells such as GC B-cells require enhanced eIF4A activity through B-cell receptor (BCR) stimulation. We found that eIF4A cap-binding activity is in fact greatly enhanced upon in vitro activation of human splenic B-cells after treatment with anti-BCR, while the pharmacological inhibitor of eIF4A, Silvestrol, potently inhibited BCR-induced protein translation. Upon antigen-induced activation, naïve, IgM+ B-cells differentiate within the germinal centers (GC) and undergo isotype switching to IgG. We found that IgG expressing human splenic B-cells displayed more robust eIF4A cap-binding activity and protein translation than IgM expressing B-cells after isotype specific BCR-stimulation. We also performed translational profiling to determine which mRNAs are preferentially loaded onto the polysome upon BCR activation and depleted upon pharmacological inhibition of eIF4A. Among the top differentially modulated genes was caspase recruitment domain family member 11 (CARD11). We found that CARD11, along with the B-cell CLL/lymphoma 10 (BCL10) and mucosa associated lymphoid tissue lymphoma translocation gene 1 (MALT1) harbor complex 5'UTRs unlike the majority of other components in the BCR signaling cascade. Importantly, these proto-oncogenes form the CARD11-BCL10-MALT1 (CBM) signalosome, which mediates BCR activation of nuclear factor of kappa light chain gene enhancer in B-cells (NF-κB) signaling. Both constitutive activation of NF-κB and the aberrant expression of CBM components are associated with B-cell lymphomas in humans. Collectively, these data indicate that eIF4A-mediated control of oncogene translation may be a critical component for B-cell lymphoma progression and suggest that pharmacological targeting of eIF4A may be an attractive therapeutic approach in the management of human B-cell lymphomas.