Directional emission of dielectric disks with a finite scatterer in the THz regime.

In the Terahertz (THz) domain, we investigate both numerically and experimentally the directional emission of whispering gallery mode resonators that are perturbed by a small scatterer in the vicinity of the resonators rim. We determine quality factor degradation, the modal structure and the emission direction for various geometries. We find that scatterers do allow for directional emission without destroying the resonator's quality factor. This finding allows for new geometries and outcoupling scenarios for active whispering gallery mode structures such as quantum cascade lasers and passive resonators such as evanescent sensors. The experimental results agree well with finite difference time domain simulations.

Effect of verapamil on the action of methanethiosulfonate reagents on human voltage-gated K(v)1.3 channels: implications for the C-type inactivated state.

BACKGROUND AND PURPOSE: Voltage-gated K(v)1.3 channels appear on T-lymphocytes and are characterized by their typical C-type inactivation. In order to develop drugs stabilizing the C-type inactivated state and thus potentially useful in treatment of autoimmune diseases, it is important to know more about the three-dimensional structure of this inactivated state of the channel. EXPERIMENTAL APPROACH: The patch-clamp technique was used to study effects of methanethiosulphonate (MTS) compounds on currents through wild-type human K(v)1.3 (hK(v)1.3) and two mutant channels, hK(v)1.3 V417C and hK(v) 1.3 H399T-V417C, in the closed, open and inactivated states. KEY RESULTS: Extracellular application of 2-aminoethyl methanethiosulphonate (MTSEA) irreversibly reduced currents through hK(v) 1.3 V417C channels in the open and inactivated, but not in the closed state, indicating that a modification was possible. Co-application of verapamil prevented this reduction. Intracellular application of MTSEA and [2-(trimethylammonium)ethyl] methanethiosulphonate (MTSET) also modified the mutant channels, whereas extra- and intracellular application of sodium (2-sulfonatoethyl)methanethiosulphonate (MTSES) and intracellular application of MTSET did not. CONCLUSIONS AND IMPLICATIONS: Our experiments showed that the binding site for MTS compounds was intracellular in the mutant channels and that the V417C mutant channels were modified in the open and the inactivated states, and this modification was prevented by verapamil. Therefore, the activation gate on the intracellular side of the selectivity filter must be open during inactivation. Furthermore, although the S6 segment is moving further apart during inactivation, this change does not include a movement of the side chain of the amino acid at position 417, away from lining the channel pore.

Repression of the integrated papillomavirus E6/E7 promoter is required for growth suppression of cervical cancer cells.

The human papillomavirus (HPV) E2 protein is an important regulator of viral E6 and E7 gene expression. E2 can repress the viral promoter for E6 and E7 expression as well as block progression of the cell cycle in cancer cells harboring the DNA of "high-risk" HPV types. Although the phenomenon of E2-mediated growth arrest of HeLa cells and other HPV-positive cancer cells has been well documented, the specific mechanism by which E2 affects cellular proliferation has not yet been elucidated. Here, we show that bovine papillomavirus (BPV) E2-induced growth arrest of HeLa cells requires the repression of the E6 and E7 promoter. This repression is specific for E2TA and not E2TR, a BPV E2 variant that lacks the N-terminal transactivation domain. We demonstrate that expression of HPV16 E6 and E7 from a heterologous promoter that is not regulated by E2 rescues HeLa cells from E2-mediated growth arrest. Our data indicate that the pathway of E2-mediated growth arrest of HeLa cells requires repression of E6 and E7 expression through an activity specified by the transactivation domain of E2TA.

Cellular components interact with adenovirus type 5 minimal DNA packaging domains.

Adenovirus type 5 DNA packaging is initiated from the left end of the viral genome and depends on the presence of a cis-acting packaging domain located between nucleotides 194 and 380. Multiple redundant packaging elements (termed A repeats I through VII [AI through AVII]) are contained within this domain and display differential abilities to support DNA packaging in vivo. The functionally most important repeats, AI, AII, AV, and AVI, follow a bipartite consensus motif exhibiting AT-rich and CG-rich core sequences. Results from previous mutational analyses defined a fragment containing AV, AVI, and AVII as a minimal packaging domain in vivo, which supports a functional independence of the respective cis-acting sequences. Here we describe multimeric versions of individual packaging elements as minimal packaging domains that can confer viability and packaging activity to viruses carrying gross truncations within their left end. These mutant viruses directly rate the functional role that different packaging elements play relative to each other. The A repeats are likely to be binding sites for limiting, trans-acting packaging factors of cellular and/or viral origin. We report here the characterization of two cellular binding activities interacting with all of the minimal packaging domains in vitro, an unknown binding activity termed P-complex, and the transcription factor chicken ovalbumin upstream promoter transcription factor. The binding of both activities is dependent on the integrity of the AT-rich, but not the CG-rich, consensus half site. In the case of P-complex, binding affinity for different minimal packaging domains in vitro correlates well with their abilities to support DNA packaging in vivo. Interestingly, P-complex interacts not only with packaging elements but also with the left terminus of the viral genome, the core origin of replication. Our data implicate cellular factors as components of the viral packaging machinery. The dual binding specificity of P-complex for packaging and replication sequences may further suggest a direct involvement of left-end replication sequences in viral DNA encapsidation.

Bipartite structure and functional independence of adenovirus type 5 packaging elements.

Selectivity and polarity of adenovirus type 5 DNA packaging are believed to be directed by an interaction of putative packaging factors with the cis-acting adenovirus packaging domain located within the genomic left end (nucleotides 194 to 380). In previous studies, this packaging domain was mutationally dissected into at least seven functional elements called A repeats. These elements, albeit redundant in function, exhibit differences in the ability to support viral packaging, with elements I, II, V, and VI as the most critical repeats. Viral packaging was shown to be sensitive to spatial changes between individual A repeats. To study the importance of spatial constraints in more detail, we performed site-directed mutagenesis of the 21-bp linker regions separating A repeats I and II, as well as A repeats V and VI. The results of our mutational analysis reveal previously unrecognized sequences that are critical for DNA encapsidation in vivo. On the basis of these results, we present a more complex consensus motif for the adenovirus packaging elements which is bipartite in structure. DNA encapsidation is compromised by changes in spacing between the two conserved parts of the consensus motif, rather than between different A repeats. Genetic evidence implicating packaging elements as independent units in viral DNA packaging is derived from the selection of revertants from a packaging-deficient adenovirus: multimerization of packaging repeats is sufficient for the evolution of packaging-competent viruses. Finally, we identify minimally sized segments of the adenovirus packaging domain that can confer viability and packaging activity to viruses carrying gross truncations within their left-end sequences. Coinfection experiments using the revertant as well as the minimal-packaging-domain mutant viruses strengthen existing arguments for the involvement of limiting, trans-acting components in viral DNA packaging.