Mechanisms for Kinase-mediated Dimerization of the Epidermal Growth Factor Receptor

We study a mechanism by which dimerization of the EGF receptor (EGFR) cytoplasmic domain is transmitted to the ectodomain. Therapeutic and other small molecule antagonists to the kinase domain that stabilize its active conformation, but not those that stabilize an inactive conformation, stabilize ectodomain dimerization. Inhibitor-induced dimerization requires an asymmetric kinase domain interface associated with activation. EGF and kinase inhibitors stimulate formation of identical dimer interfaces in the EGFR transmembrane domain, as shown by disulfide cross-linking. Disulfide cross-linking at an interface in domain IV in the ectodomain was also stimulated similarly; however, EGF but not inhibitors stimulated cross-linking in domain II. Inhibitors similarly induced noncovalent dimerization in nearly full-length, detergent-solubilized EGFR as shown by gel filtration. EGFR ectodomain deletion resulted in spontaneous dimerization, whereas deletion of exons 2–7, in which extracellular domains III and IV are retained, did not. In EM, kinase inhibitor-induced dimers lacked any well defined orientation between the ectodomain monomers. Fab of the therapeutic antibody cetuximab to domain III confirmed a variable position and orientation of this domain in inhibitor-induced dimers but suggested that the C termini of domain IV of the two monomers were in close proximity, consistent with dimerization in the transmembrane domains. The results provide insights into the relative energetics of intracellular and extracellular dimerization in EGFR and have significance for physiologic dimerization through the asymmetric kinase interface, bidirectional signal transmission in EGFR, and mechanism of action of therapeutics.     

Immunoglobulin Fc fragment tagging allows strong activation of endogenous CD4 T cells to reshape the tumor milieu and enhance the antitumor effect of lentivector immunization

A major problem with current cancer vaccines is that the induction of CD8 immune responses is rarely associated with antitumor benefits, mainly owing to multiple immune suppressions in established tumor lesions. In this study, we investigated if and how activation of endogenous CD4 T cells could be achieved to influence the suppressive tumor milieu and antitumor effect. We engineered a lentivector (lv) to express a nominal fusion Ag composed of hepatitis B surface protein and IgG2a Fc fragment (HBS-Fc-lv) to increase the magnitude of CD8 response but, more importantly, to induce effective coactivation of CD4 T cells. We found that, remarkably, immunization with HBS-Fc-lv caused significant regression of established tumors. Immunologic analysis revealed that, compared with HBS-lv without Fc fragment, immunization with HBS-Fc-lv markedly increased the number of functional CD8 and CD4 T cells and the level of Th1/Tc1-like cytokines in the tumor while substantially decreasing the regulatory T cell ratio. The favorable immunologic changes in tumor lesions and the improvement of antitumor effects from HBS-Fc-lv immunization were dependent on the CD4 activation, which was Fc receptor mediated. Adoptive transfer of CD4 T cells from the HBS-Fc-lv-immunized mice could activate endogenous CD8 T cells in an IFN-γ-dependent manner. We conclude that endogenous CD4 T cells can be activated by lv expressing Fc-tagged Ag to provide another layer of help--that is, creating a Th1/Tc1-like proinflammatory milieu within the tumor lesion to boost the effector phase of immune responses in enhancing the antitumor effect.     

Complete Genome Sequence of IME13, a Stenotrophomonas maltophilia Bacteriophage with Large Burst Size and Unique Plaque Polymorphism

Stenotrophomonas maltophilia bacteriophage IME13 is a virulent phage with a large burst size, exceeding 3,000, much larger than that of any other stenotrophomonas phage reported before. It showed effective lysis of Stenotrophomonas maltophilia. Additionally, the phage IME13 developed at least three obviously different sizes of plaques when a single plaque was picked out and inoculated on a double-layer Luria broth agar plate with its host. Here we announce its complete genome and describe major findings from its annotation.     

Replicating adenovirus-simian immunodeficiency virus (SIV) recombinant priming and envelope protein boosting elicits localized, mucosal IgA immunity in rhesus macaques correlated with delayed acquisition following a repeated low-dose rectal SIV(mac251) challenge

We have shown that sequential replicating adenovirus type 5 host range mutant human immunodeficiency virus/simian immunodeficiency virus (HIV/SIV) recombinant priming delivered first intranasally (i.n.) plus orally and then intratracheally (i.t.), followed by envelope protein boosting, elicits broad cellular immunity and functional, envelope-specific serum and mucosal antibodies that correlate with protection from high-dose SIV and simian/human immunodeficiency virus (SHIV) challenges in rhesus macaques. Here we extended these studies to compare the standard i.n./i.t. regimen with additional mucosal administration routes, including sublingual, rectal, and vaginal routes. Similar systemic cellular and humoral immunity was elicited by all immunization routes. Central and effector memory T cell responses were also elicited by the four immunization routes in bronchoalveolar lavage fluid and jejunal, rectal, and vaginal tissue samples. Cellular responses in vaginal tissue were more compartmentalized, being induced primarily by intravaginal administration. In contrast, all immunization routes elicited secretory IgA (sIgA) responses at multiple mucosal sites. Following a repeated low-dose intrarectal (i.r.) challenge with SIV(mac251) at a dose transmitting one or two variants, protection against acquisition was not achieved except in one macaque in the i.r. immunized group. All immunized macaques exhibited reduced peak viremia compared to that of controls, correlated inversely with prechallenge serum antienvelope avidity, antibody-dependent cellular cytotoxicity (ADCC) titers, and percent antibody-dependent cell-mediated viral inhibition. Both antibody avidity and ADCC titers were correlated with the number of exposures required for infection. Notably, we show for the first time a significant correlation of vaccine-induced sIgA titers in rectal secretions with delayed acquisition. Further investigation of the characteristics and properties of the sIgA should elucidate the mechanism leading to this protective effect.     

Comparative morphology of minute intestinal fluke eggs that can occur in human stools in the republic of Korea

The egg morphology of minute intestinal flukes (MIF) that can occur as human infections in the Republic of Korea, i.e., Metagonimus yokogawai, M. miyatai, M. takahashii, Heterophyes nocens, Heterophyopsis continua, Stellantchasmus falcatus, Stictodora fuscata, Pygidiopsis summa, and Gymnophalloides seoi, was studied in comparison with Clonorchis sinensis. The adult worms were obtained from residents of endemic areas, and their intrauterine eggs were studied and measured using light microscopy; the length, width, length-width ratio (LWR), and Faust-Meleney index (FMI). Several specimens were processed for scanning electron microscopy (SEM), and before gold-coating, the uterine portion of each fluke was etched with a sharp pin in order to expose the eggs. The MIF eggs were ovoid, pyriform, or elliptical with a size range of 21-35×12-21 μm. S. fuscata eggs revealed the highest FMI (largest in the area) and lowest LWR, whereas P. summa eggs showed the lowest FMI and medium LWR. SEM revealed that G. seoi and S. fuscata had remarkably clean shell surface lacking the muskmelon-like structure which is prominent in C. sinensis eggs. In Metagonimus spp., H. continua, H. nocens, and S. falcatus eggs, minute surface ridges were recognizable though less prominent compared with C. sinensis. On the surface of P. summa eggs, thread-like curly structures were characteristically seen. The results revealed that important differential keys for MIF eggs include the length, width, area (FMI), shape of the eggs, and the extent of the muskmelon-like structure or ridges on their shell surface and operculum.     

The alkenyl migration mechanism catalyzed by extradiol dioxygenases: a hybrid DFT study

6-Hydroxymethyl-6-methylcyclohexa-2,4-dienone is a mechanistic probe which when incubated with an extradiol dioxygenase yields a 2-tropolone product. This observation was originally interpreted as evidence supporting a direct heterolytic 1,2-alkenyl migration mechanism for a ring expansion reaction catalyzed by this class of Fe(II)-dependent nonheme enzymes (Xin and Bugg in J Am Chem Soc 130:10422-10430, 2008). In the work reported in this contribution we used quantum chemical methods to test whether such a mechanism is energetically possible and we found that it is not, neither for the mechanistic probe nor for the native catalytic cycle intermediate. Models of increasing complexity were used to calculate energy barriers to the heterolytic 1,2-alkenyl migration and alternative radical mechanisms. It was found that the former involves substantially higher barriers than the latter. A tentative radical mechanism that accounts for the transformation of the probe substrate to 2-tropolone was also proposed, and it involves acceptable barriers.     

DNAzyme-functionalized Pt nanoparticles/carbon nanotubes for amplified sandwich electrochemical DNA analysis

A novel DNAzyme-functionalized Pt nanoparticles/carbon nanotubes (DNAzyme/Pt NPs/CNTs) bioconjugate was fabricated as trace tag for ultrasensitive sandwich DNA detection. The Pt NPs/CNTs were prepared via layer-by-layer (LBL) assembly of the Pt NPs and polyelectrolyte on the carboxylated CNTs, followed by the functionalization with the DNAzyme and reporter probe DNA through the platinum-sulfur bonding. The subsequent sandwich-type DNA specific reaction would confine numerous DNAzyme/Pt NPs/CNTs bioconjugate onto the gold electrode surface for amplifying the signal. In the presence of 3,3',5,5' tetramethylbenzidine (TMB) which could be oxidized by the DNAzyme, electrochemical signals could be generated by chronoamperometry via the interrogation of reduction electrochemical signal of oxidized TMB. The constructed DNA sensor exhibited a wide linear response to target DNA ranging from 1.0fM to 10pM with the detection limit down to 0.6fM and exhibited excellent selectivity against even a single base mismatch. In addition, this novel DNA sensor showed fairly good reproducibility, stability, and reusability.     

A new pancreatic lipase inhibitor from Broussonetia kanzinoki

A new phenolic compound, broussonone A (1) were isolated from the stem barks of Broussonetia kanzinoki (Moraceae), together with two diphenylpropanes, broussonin A (2), broussonin B (3), two flavans, 7,4'-dihydroxyflavan (4), 3',7-dihydroxy-4'-methoxyflavan (5), and two flavones, 3,7-dihydroxy-4'-methoxyflavone (6), 3,7,3'-trihydroxy-4'-methoxyflavone (7). Compound 1 showed noncompetitive inhibitory activity on pancreatic lipase with an IC(50) of 28.4 μM. In addition, compounds 1-5 significantly inhibited adipocyte differentiation in 3T3-L1 cells as measured fat accumulation using Oil Red O assay.     

Crystal structure and biochemical analyses reveal Beclin 1 as a novel membrane binding protein

The Beclin 1 gene is a haplo-insufficient tumor suppressor and plays an essential role in autophagy. However, the molecular mechanism by which Beclin 1 functions remains largely unknown. Here we report the crystal structure of the evolutionarily conserved domain (ECD) of Beclin 1 at 1.6 Å resolution. Beclin 1 ECD exhibits a previously unreported fold, with three structural repeats arranged symmetrically around a central axis. Beclin 1 ECD defines a novel class of membrane-binding domain, with a strong preference for lipid membrane enriched with cardiolipin. The tip of a surface loop in Beclin 1 ECD, comprising three aromatic amino acids, acts as a hydrophobic finger to associate with lipid membrane, consequently resulting in the deformation of membrane and liposomes. Mutation of these aromatic residues rendered Beclin 1 unable to stably associate with lipid membrane in vitro and unable to fully rescue autophagy in Beclin 1-knockdown cells in vivo. These observations form an important framework for deciphering the biological functions of Beclin 1.