Lidocaine block of cardiac sodium channels

Lidocaine block of cardiac sodium channels was studied in voltage-clamped rabbit purkinje fibers at drug concentrations ranging from 1 mM down to effective antiarrhythmic doses (5-20 μM). Dose-response curves indicated that lidocaine blocks the channel by binding one-to-one, with a voltage-dependent K(d). The half-blocking concentration varied from more than 300 μM, at a negative holding potential where inactivation was completely removed, to approximately 10 μM, at a depolarized holding potential where inactivation was nearly complete. Lidocaine block showed prominent use dependence with trains of depolarizing pulses from a negative holding potential. During the interval between pulses, repriming of I (Na) displayed two exponential components, a normally recovering component (τless than 0.2 s), and a lidocaine-induced, slowly recovering fraction (τ approximately 1-2 s at pH 7.0). Raising the lidocaine concentration magnified the slowly recovering fraction without changing its time course; after a long depolarization, this fraction was one-half at approximately 10 μM lidocaine, just as expected if it corresponded to drug-bound, inactivated channels. At less than or equal to 20 μM lidocaine, the slowly recovering fraction grew exponentially to a steady level as the preceding depolarization was prolonged; the time course was the same for strong or weak depolarizations, that is, with or without significant activation of I(Na). This argues that use dependence at therapeutic levels reflects block of inactivated channels, rather than block of open channels. Overall, these results provide direct evidence for the “modulated-receptor hypothesis” of Hille (1977) and Hondeghem and Katzung (1977). Unlike tetrodotoxin, lidocaine shows similar interactions with Na channels of heart, nerve, and skeletal muscle.     

Interstitial 3p deletion in a child due to paternal paracentric inserted inversion.

An infant with multiple anomalies and developmental delay during his first year was found to have an intersitital deletion of band p14 from the proximal short arm of chromosome 3. Examination of the father's chromosomes indicates an "inserted paracentric inversion" in chromosome 3 as the probable origin of the deletion in the child.     

Cyto-immunological studies of guinea pig sperm antigens

Summary Antigenic localization in guinea pig epididymal sperm and testicular imprints as well as in viable, motile guinea pig epididymal sperm was studied by means of fluorescent labelled antibody techniques. Globulins from rabbits and chickens immunized with guinea pig epididymal sperm were used in the direct procedure while sera from sheep and fowl injected with rabbit globulins were used in the indirect procedure. The main findings were: 1) spermatozoa from the distal portion of the epididymis displayed brilliant fluorescent acrosomes and less intensely stained midpieces and principal pieces when treated as dried smears in both the direct and indirect methods; 2) testicular spermatozoa were similarly stained but whereas in epididymal spermatozoa the whole acrosome stained intensely, the testicular spermatozoal acrosome displayed intense fluorescence of the inner acrosome; 3) protoplasmic droplets fluoresced strongly; 4) cross-reactivity was observed between human and guinea pig sperm but not between rat and guinea pig sperm, indicating an antigenic relationship between human and guinea pig but not between guinea pig and rat; 5) treatment of viable, motile guinea pig spermatozoa with fluorescent globulins resulted in agglutination and immobilization as well as formation of antigen-antibody aggregates adherent to the cell membrane of the head, midpiece and principal piece; the formation of such fluorescent aggregates in the medium surrounding the treated motile sperm was indicative of leaching of antigenic material from the sperm cells.This investigation was supported by funds from United States Public Health Service grant HE-05798-03, The Ford Foundation and National Science Foundation.     

Associations between mites and leaf dornatia

Associations between mites and leaf domatia are widespread, abundant and probably ancient. Recent research has shown that mites commonly shelter, develop and reproduce within domatia on plants in many geographic regions, from the tropics to the temperate zone, and most of these mites belong to predaceous or fungivorous taxa. Of hypotheses offered to explain these associations, protective mutualism is most consistent with the reported characteristics and patterns of mitedomatia association.     

Salt links dominate affinity of antibody HyHEL-5 for lysozyme through enthalpic contributions.

The binding of murine monoclonal antibody HyHEL-5 to lysozyme has been the subject of extensive crystallographic, computational, and experimental investigations. The complex of HyHEL-5 with hen egg lysozyme (HEL) features salt bridges between Fab heavy chain residue Glu(50), and Arg(45) and Arg(68) of HEL. This interaction has been predicted to play a dominant role in the association on the basis of molecular electrostatics calculations. The association of aspartic acid and glutamine mutants at position 50(H) of the cloned HyHEL-5 Fab with HEL and bobwhite quail lysozyme (BQL), an avian variant bearing an Arg(68) --> Lys substitution in the epitope, was characterized by isothermal titration calorimetry and sedimentation equilibrium. Affinities for HEL were reduced by 400-fold (E50(H)D) and 40,000-fold (E50(H)Q) (DeltaDeltaG degrees estimated at 4.0 and 6.4 kcal mol(-1), respectively). The same mutations reduce affinity for BQL by only 7- and 55-fold, respectively, indicating a reduced specificity for HEL. The loss of affinity upon mutation is in each case primarily due to an unfavorable change in the enthalpy of the interaction; the entropic contribution is virtually unchanged. An enthalpy-entropy compensation exists for each interaction; DeltaH degrees decreases, while DeltaS degrees increases with temperature. The DeltaCp for each mutant interaction is less negative than the wild-type. Mutant-cycle analysis suggests the mutations present in the HyHEL-5 Fab mutants are linked to those present in the BQL with coupling energies between 3 and 4 kcal mol(-1).     

DNAzyme-mediated recovery of small recombinant RNAs from a 5S rRNA-derived chimera expressed in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Background  

Manufacturing large quantities of recombinant RNAs by overexpression in a bacterial host is hampered by their instability in intracellular environment. To overcome this problem, an RNA of interest can be fused into a stable bacterial RNA for the resulting chimeric construct to accumulate in the cytoplasm to a sufficiently high level. Being supplemented with cost-effective procedures for isolation of the chimera from cells and recovery of the recombinant RNA from stabilizing scaffold, this strategy might become a viable alternative to the existing methods of chemical or enzymatic RNA synthesis.     

Grb7 SH2 domain structure and interactions with a cyclic peptide inhibitor of cancer cell migration and proliferation

Background

Human growth factor receptor bound protein 7 (Grb7) is an adapter protein that mediates the coupling of tyrosine kinases with their downstream signaling pathways. Grb7 is frequently overexpressed in invasive and metastatic human cancers and is implicated in cancer progression via its interaction with the ErbB2 receptor and focal adhesion kinase (FAK) that play critical roles in cell proliferation and migration. It is thus a prime target for the development of novel anti-cancer therapies. Recently, an inhibitory peptide (G7-18NATE) has been developed which binds specifically to the Grb7 SH2 domain and is able to attenuate cancer cell proliferation and migration in various cancer cell lines.

Results

As a first step towards understanding how Grb7 may be inhibited by G7-18NATE, we solved the crystal structure of the Grb7 SH2 domain to 2.1 Å resolution. We describe the details of the peptide binding site underlying target specificity, as well as the dimer interface of Grb 7 SH2. Dimer formation of Grb7 was determined to be in the μM range using analytical ultracentrifugation for both full-length Grb7 and the SH2 domain alone, suggesting the SH2 domain forms the basis of a physiological dimer. ITC measurements of the interaction of the G7-18NATE peptide with the Grb7 SH2 domain revealed that it binds with a binding affinity of K_d = ~35.7 μM and NMR spectroscopy titration experiments revealed that peptide binding causes perturbations to both the ligand binding surface of the Grb7 SH2 domain as well as to the dimer interface, suggesting that dimerisation of Grb7 is impacted on by peptide binding.

Conclusion

Together the data allow us to propose a model of the Grb7 SH2 domain/G7-18NATE interaction and to rationalize the basis for the observed binding specificity and affinity. We propose that the current study will assist with the development of second generation Grb7 SH2 domain inhibitors, potentially leading to novel inhibitors of cancer cell migration and invasion.     

X-ray crystal structures of the estrogen-related receptor-gamma ligand binding domain in three functional states reveal the molecular basis of small molecule regulation

X-ray crystal structures of the ligand binding domain (LBD) of the estrogen-related receptor-gamma (ERRgamma) were determined that describe this receptor in three distinct states: unliganded, inverse agonist bound, and agonist bound. Two structures were solved for the unliganded state, the ERRgamma LBD alone, and in complex with a coregulator peptide representing a portion of receptor interacting protein 140 (RIP140). No significant differences were seen between these structures that both exhibited the conformation of ERRgamma seen in studies with other coactivators. Two structures were obtained describing the inverse agonist-bound state, the ERRgamma LBD with 4-hydroxytamoxifen (4-OHT), and the ERRgamma LBD with 4-OHT and a peptide representing a portion of the silencing mediator of retinoid and thyroid hormone action protein (SMRT). The 4-OHT structure was similar to other reported inverse agonist bound structures, showing reorientation of phenylalanine 435 and a displacement of the AF-2 helix relative to the unliganded structures with little other rearrangement occurring. No significant changes to the LBD appear to be induced by peptide binding with the addition of the SMRT peptide to the ERRgamma plus 4-OHT complex. The observed agonist-bound state contains the ERRgamma LBD, a ligand (GSK4716), and the RIP140 peptide and reveals an unexpected rearrangement of the phenol-binding residues. Thermal stability studies show that agonist binding leads to global stabilization of the ligand binding domain. In contrast to the conventional mechanism of nuclear receptor ligand activation, activation of ERRgamma by GSK4716 does not appear to involve a major rearrangement or significant stabilization of the C-terminal helix.