Analysis of the 3(')-hydroxyl group in Drosophila siRNA function

Members of the RNA-dependent RNA polymerase (RdRP) gene family have been shown to be essential for dsRNA-mediated gene silencing based on genetic screens in a variety of organisms, including Caenorhabditis elegans, Arabidopsis, Neurospora, and Dictyostelium. A hallmark of this process is the formation of small 21- to 25-bp dsRNAs, termed siRNAs for small interfering RNAs, which are derived from the dsRNA that initiates gene silencing. We have developed methods to demonstrate that these siRNAs produced in Drosophila embryo extract can be uniformly incorporated into dsRNA in a template-specific manner that is subsequently degraded by RNase III-related enzyme activity to create a second generation of siRNAs. SiRNA function in dsRNA synthesis and mRNA degradation depends upon the integrity of the 3'-hydroxyl of the siRNA, consistent with the interpretation that siRNAs serve as primers for RdRP activity in the formation of dsRNA. This process of siRNA incorporation into dsRNA followed by degradation and the formation of new siRNAs has been termed "degradative PCR" and the proposed mechanism is consistent with the genetic and biochemical data derived from studies in C. elegans, Arabidopsis, Drosophila, and Dictyostelium. The methods used to study the function of both natural and synthetic siRNAs in RNA interference in Drosophila embryo extracts are detailed. The importance of the 3'-hydroxyl group for siRNA function and its incorporation into dsRNA is emphasized and the results support a model that places RNA-dependent RNA polymerase as a key mediator in the RNA interference mechanism in Drosophila.     

Nuclear ribosomal DNA sequence polymorphism and hybridization in checker mallows (Sidalcea, Malvaceae)

Checker mallows (Sidalcea, Malvaceae) constitute a western North American genus of annuals and perennials that have been regarded as taxonomically difficult because of complex patterns of morphological variation putatively stemming from hybridization and polyploidy. In recent molecular phylogenetic investigations extensive polymorphism was observed in the internal and external transcribed spacers (ITS and ETS) of 18S-26S nuclear ribosomal DNA for some Sidalcea samples. To resolve the evolutionary basis for this polymorphism and to readdress the evolutionary impact of hybridization in Sidalcea we cloned and sequenced the polymorphic DNAs and included the clones in phylogenetic analyses together with direct sequences of non-polymorphic samples. The positions of cloned spacer sequences in the phylogenetic trees suggest that S. reptans and two subspecies of S. malviflora may have been influenced by past hybridization with lineages of the "glaucescens" clade. Polymorphic sequence patterns in other taxa may be a result of extensive interbreeding within young clades, in keeping with the minimal sequence divergence, largely overlapping geographic distributions and morphology, and ploidy variation in these groups. Other possible explanations for polymorphic sequences in members of Sidalcea include slow concerted evolution relative to mutation rates, incomplete lineage sorting, and recent pseudogene formation.     

Structural basis of membrane binding by Gla domains of vitamin K-dependent proteins

In a calcium-dependent interaction critical for blood coagulation, vitamin K-dependent blood coagulation proteins bind cell membranes containing phosphatidylserine via gamma-carboxyglutamic acid-rich (Gla) domains. Gla domain-mediated protein-membrane interaction is required for generation of thrombin, the terminal enzyme in the coagulation cascade, on a physiologic time scale. We determined by X-ray crystallography and NMR spectroscopy the lysophosphatidylserine-binding site in the bovine prothrombin Gla domain. The serine head group binds Gla domain-bound calcium ions and Gla residues 17 and 21, fixed elements of the Gla domain fold, predicting the structural basis for phosphatidylserine specificity among Gla domains. Gla domains provide a unique mechanism for protein-phospholipid membrane interaction. Increasingly Gla domains are being identified in proteins unrelated to blood coagulation. Thus, this membrane-binding mechanism may be important in other physiologic processes.     

Immunopathogenesis and immunotherapy in AIDS virus infections

The heterogeneity of HIV and the different human leukocyte antigen (HLA) backgrounds of infected individuals have posed challenges to understanding the pathogenesis of HIV infection. But continuing advances in our knowledge of the role of immune responses in controlling HIV viremia should help to define goals for immune-based therapies and vaccine strategies against AIDS.     

Low power millimeter wave irradiation exerts no harmful effect on human keratinocytes in vitro

Low power millimeter wave (LP-MW) irradiation has been successfully used in clinical practice as an independent and/or supplemental therapy in patients with various diseases. It is still not clear, however, whether exposed skin is directly affected by repeated LP-MW irradiation and whether cells of the epidermis can be activated by the absorbed energy. Keratinocytes, the most numerous component of the epidermis are believed to manifest functional responses to physical stimuli. In this study we analyzed whether LP-MW irradiation modulated the production of chemokines, including RANTES and IP-10 of keratinocytes in vitro. We also investigated whether LP-MW irradiation induces a heat stress reaction in keratinocytes, and stimulates heat shock protein 70 (Hsp70) production. Vital staining of keratinocytes with carboxyfluorescein succinimidyl ester and ethidium bromide was used to analyze the MW effect on the viability of adherent cells. In addition, we studied the effect of LP-MW irradiation on intercellular gap junctional communication in keratinocyte monolayers by Lucifer yellow dye transfer. We found no significant changes in constitutive RANTES and inducible IP-10 production following LP-MW irradiation. LP-MW exposure of keratinocyte monolayers did not alter Hsp70 production, unlike exposure to higher power MWs (HP-MW) or hyperthermia (43 degrees C; 1 h). LP-MW irradiation and hyperthermia did not alter the viability of adherent keratinocytes, while HP-MW irradiation induced cellular damage within the beam area. Finally, we found no alteration in the gap junctional intercellular communication of keratinocytes following LP-MW irradiation, which on the other hand, was significantly increased by hyperthermia. In summary, we detected no harmful effect of LP-MW irradiation on both keratinocyte function and structure in vitro, although these cells were sensitive to higher MW power that developed heat stress reaction and cellular damage. Our results provide further evidence that LP-MW irradiation does not induce evidence of skin inflammation or keratinocyte damage and that its clinical application appears to be safe.