Trypanosome U-deletional RNA editing involves guide RNA-directed endonuclease cleavage, terminal U exonuclease, and RNA ligase activities.

We have studied the mechanism of accurate in vitro RNA editing of Trypanosoma brucei ATPase 6 mRNA, using four mRNA-guide RNA (gRNA) pairs that specify deletion of 2, 3, or 4 U residues at editing site 1 and mitochondrial extract. This extract not only catalyzes deletion of the specified number of U residues but also exhibits a novel endonuclease activity that cleaves the input pre-mRNA in a gRNA-directed manner, precisely at the phosphodiester bond predicted in a simple enzymatic model of RNA editing. This cleavage site is inconsistent with a chimera-based editing mechanism. The U residues to be deleted, present at the 3' end of the upstream cleavage product, are then removed evidently by a 3' U-specific exonuclease and not by a reverse reaction of terminal U transferase. RNA ligase can then join the mRNA halves through their newly formed 5' P and 3' OH termini, generating mRNA faithfully edited at the first editing site. This resultant, partially edited mRNA can then undergo accurate, gRNA-directed cleavage at editing site 2, again precisely as predicted by the enzymatic editing model. All of these enzymatic activities cofractionate with the U-deletion activity and may reside in a single complex. The data imply that each round of editing is a four-step process, involving (i) gRNA-directed cleavage of the pre-mRNA at the bond immediately 5' of the region base paired to the gRNA, (ii) U deletion from or U addition to the 3' OH of the upstream mRNA half, (iii) ligation of the mRNA halves, and (iv) formation of additional base pairing between the correctly edited site and the gRNA that directs subsequent nuclease cleavage at the next editing site.

Second generation hybrid polar compounds are potent inducers of transformed cell differentiation.

Hybrid polar compounds, of which hexamethylenebisacetamide (HMBA) is the prototype, are potent inducers of differentiation of murine erythroleukemia (MEL) cells and a wide variety of other transformed cells. HMBA has been shown to induce differentiation of neoplastic cells in patients, but is not an adequate therapeutic agent because of dose-limiting toxicity. We report on a group of three potent second generation hybrid polar compounds, diethyl bis-(pentamethylene-N,N-dimethylcarboxamide) malonate (EMBA), suberoylanilide hydroxamic acid (SAHA), and m-carboxycinnamic acid bis-hydroxamide (CBHA) with optimal concentrations for inducing MEL cells of 0.4 mM, 2 microM, and 4 microM, respectively, compared to 5 mM for HMBA. All three agents induce accumulation of underphosphorylated pRB; increased levels of p2l protein, a prolongation of the initial G1 phase of the cell cycle; and accumulation of hemoglobin. However, based upon their effective concentrations, the cross-resistance or sensitivity of an HMBA-resistant MEL cell variant, and differences in c-myb expression during induction, these differentiation-inducing hybrid polar compounds can be grouped into two subsets, HMBA/EMBA and SAHA/CBHA. This classification may prove of value in selecting and planning prospective preclinical and clinical studies toward the treatment of cancer by differentiation therapy.

Oligodeoxynucleotides inhibit retinal neovascularization in a murine model of proliferative retinopathy.

Diseases characterized by retinal neovascularization are among the principal causes of visual loss worldwide. The hypoxia-stimulated expression of vascular endothelial growth factor (VEGF) has been implicated in the proliferation of new blood vessels. We have investigated the use of antisense phosphorothioate oligodeoxynucleotides against murine VEGF to inhibit retinal neovascularization and VEGF synthesis in a murine model of proliferative retinopathy. Intravitreal injections of two different antisense phosphorothioate oligodeoxynucleotides prior to the onset of proliferative retinopathy reduced new blood vessel growth a mean of 25 and 31% compared with controls. This inhibition was dependent on the concentration of antisense phosphorothioate oligodeoxynucleotides and resulted in a 40-66% reduction in the level of VEGF protein, as determined by Western blot analysis. Control (sense, nonspecific) phosphorothioate oligodeoxynucleotides did not cause a significant reduction in retinal neovascularization or VEGF protein levels. These data further establish a fundamental role for VEGF expression in ischemia-induced proliferative retinopathies and a potential therapeutic use for antisense phosphorothioate oligodeoxynucleotides.

Potent immunogenicity of the B subunits of Escherichia coli heat-labile enterotoxin: receptor binding is essential and induces differential modulation of lymphocyte subsets.

The importance of receptor binding in the potent immunogenicity of Escherichia coli heat-labile enterotoxin B subunit (EtxB) was tested by comparing its immunogical properties with those of a receptor binding mutant, EtxB(G33D). Subcutaneous immunization of EtxB(G33D) resulted in 160-fold reduction in antibody titer compared with wild-type EtxB, whereas its oral delivery failed to provoke any detectable secretory or serum anti-B subunit responses. Moreover, the two proteins induced strikingly different effects on lymphocyte cultures in vitro. EtxB, in comparison with EtxB(G33D), caused an increase in the proportion of B cells, many of which were activated (CD25+); the complete depletion of CD8+ T cells; an increase in the activation of CD4+ T cells; and an increase in interleukin 2 and a decrease in interferon gamma. These data indicate that EtxB exerts profound effects on immune cells, suggesting that its potent immunogenicity is dependent not only on efficient receptor-mediated uptake, but also on direct receptor-mediated immunomodulation of lymphocyte subsets.

Rapid mass spectrometric peptide sequencing and mass matching for characterization of human melanoma proteins isolated by two-dimensional PAGE.

We report a general mass spectrometric approach for the rapid identification and characterization of proteins isolated by preparative two-dimensional polyacrylamide gel electrophoresis. This method possesses the inherent power to detect and structurally characterize covalent modifications. Absolute sensitivities of matrix-assisted laser desorption ionization and high-energy collision-induced dissociation tandem mass spectrometry are exploited to determine the mass and sequence of subpicomole sample quantities of tryptic peptides. These data permit mass matching and sequence homology searching of computerized peptide mass and protein sequence data bases for known proteins and design of oligonucleotide probes for cloning unknown proteins. We have identified 11 proteins in lysates of human A375 melanoma cells, including: alpha-enolase, cytokeratin, stathmin, protein disulfide isomerase, tropomyosin, Cu/Zn superoxide dismutase, nucleoside diphosphate kinase A, galaptin, and triosephosphate isomerase. We have characterized several posttranslational modifications and chemical modifications that may result from electrophoresis or subsequent sample processing steps. Detection of comigrating and covalently modified proteins illustrates the necessity of peptide sequencing and the advantages of tandem mass spectrometry to reliably and unambiguously establish the identity of each protein. This technology paves the way for studies of cell-type dependent gene expression and studies of large suites of cellular proteins with unprecedented speed and rigor to provide information complementary to the ongoing Human Genome Project.

Understanding the Leadership Capacity and Practice of Assistant Principals

The purpose of this study was to explore the leadership capacities and practices of assistant principals. The research also sought to determine what relationships existed between capacity and practice and to see if there was a difference based on experience, context and personal characteristics. Since the majority of principals first serve as assistant principals, their work and experiences as assistant principals will have significant consequences (Kwan, 2009). The literature has long held and continues to challenge the notion that the role of assistant principal is adequate preparation for the principalship (Chan, Webb, & Bowen, 2003; Harris, Muijs, & Crawford, 2003; Kwan, 2009; Mertz, 2000; Webb & Vulliamy, 1995). Based on empirical findings, this study has affirmed the need to further research and refine the role of the assistant principal. The results indicate that in addition to strengths, there are explicit gaps and missed opportunities in the leadership practices of assistant principals that impact the potential for building a leadership pipeline within schools. The work of the assistant principal is characterized by a proliferation of duties rather than a strategic set of practices that support distributed leadership and sustainability.

Ethnobotanical study of the sages used in traditional Valencian medicine and as essential oil: characterization of an endemic Salvia and its contribution to local development

Seven wild and cultivated Salvia species and two Phlomis species, used traditionally in Valencian medicine to treat a variety of external and internal ailments, were studied. New ethnobotanical data are provided, obtained from semistructured interviews with 34 people in the Valencian area. A seasonal characterization of the essential oil of a wild sage, Salvia blancoana Webb & Heldr. subsp. mariolensis Figuerola, by GC-FID and GC-MS was carried out as a means to ensure quality control of endemic traditional species such as this one, which has been commercialized by local industries. A comparison with the essential oil of Salvia lavandulifolia Vahl subsp.lavandulifolia allowed inclusion of the wild sage within the commercial 'Spanish sage' oil.

An ethnopharmacological and historical analysis of “Dictamnus”, a European traditional herbal medicine

Ethnopharmacological relevance and background: “Dictamnus” was a popular name for a group of medicinal herbaceous plant species of the Rutaceae and Lamiaceae, which since the 4th century have been used for gynaecological problems and other illnesses BCE and still appear in numerous ethnobotanical records. Aims: This research has as four overarching aims: Determining the historical evolution of medical preparations labelled “Dictamnus” and the different factors affecting this long-standing herbal tradition. Deciphering and differentiating those medicinal uses of “Dictamnus” which strictly correspond to Dictamnus (Rutaceae), from those of Origanum dictamnus and other Lamiaceae species. Quantitatively assessing the dependence from herbal books, and pharmaceutical tradition, of modern Dictamnus ethnobotanical records. Determining whether differences between Western and Eastern Europe exist with regards to the Dictamnus albus uses in ethnopharmacology and ethnomedicine. Methods: An exhaustive review of herbals, classical pharmacopoeias, ethnobotanical and ethnopharmacological literature was conducted. Systematic analysis of uses reported which were standardized according to International Classification of Diseases – 10 and multivariate analysis using factorial, hierarchical and neighbour joining methods was undertaken. Results and discussion: The popular concept “Dictamnus” includes Origanum dictamnus L., Ballota pseudodictamnus (L.) Benth. and B. acetabulosa (L.) Benth. (Lamiaceae), as well as Dictamnus albus L. and D. hispanicus Webb ex Willk. (Rutaceae), with 86 different types of uses. Between 1000 and 1700 CE numerous complex preparations with “Dictamnus” were used in the treatment of 35 different pathologies. On biogeographical grounds the widespread D. albus is a far more likely prototypical “Dictamnus” than the Cretan endemic Origanum dictamnus. However both form integral parts of the “Dictamnus” complex. Evidence exists for a sufficiently long and coherent tradition for D. albus and D. hispanicus, use to treat 47 different categories of diseases. Conclusions: This approach is a model for understanding the cultural history of plants and their role as resources for health care. “Dictamnus” shows how transmission of traditional knowledge about materia medica, over 26 centuries, represents remarkable levels of development and innovation. All this lead us to call attention to D. albus and D. hispanicus which are highly promising as potential herbal drug leads. The next steps of research should be to systematically analyse phytochemical, pharmacological and clinical evidence and to develop safety, pharmacology and toxicology profiles of the traditional preparations.

Declaration signed by Harvard students to not speak in the vernacular for one year, 1712 August 23

The declaration, handwritten in Latin and signed by members of the junior and sophomore classes (Harvard Classes of 1714 and 1715), promises that the undersigned will not use the vernacular but instead "whenever, at meals, at banquets,...in our rooms, in all our gatherings, wherever and whenever" will speak in Latin, Greek, or Hebrew through the next May. Additional Latin text appears on both the front and back of the document. The original is accompanied by a typed transcription and two partial handwritten translations. Note at top of original: "Script. Leonardo Dowding, Composit. a Tho. Foxcroft."

Letters from Daniel Bates to William Jenks, May 1795-September 1798

Daniel Bates wrote these five letters to his friend and classmate, William Jenks, between May 1795 and September 1798. In a letter written May 12, 1795, Bates informs Jenks, who was then employed as an usher at Mr. Webb's school, of his studies of Euclid, the meeting of several undergraduate societies, and various sightings of birds, gardens and trees. In a letter written in November 1795 from Princeton, where he was apparently on vacation with the family of classmate Leonard Jarvis, he describes playing the game "break the Pope's neck" and tells Jenks what he was reading (Nicholson, Paley?, and Thompson) and what his friend's father was reading (Mirabeau and Neckar).

Letters from John Hubbard Church to William Jenks, 1795-1798

John Hubbard Church wrote these twelve letters to his friend and classmate William Jenks between 1795 and 1798. Church wrote the letters from Boston, Rutland, Cambridge, and Chatham in Massachusetts and from Somers, Connecticut; they were sent to Jenks in Cambridge and Boston, where for a time he worked as an usher in Mr. Vinall's school and Mr. Webb's school. Church's letters touch on various subjects, ranging from his increased interest in theology and his theological studies under Charles Backus to his seasickness during a sailing voyage to Cape Cod. Church also informs Jenks of what he is reading, including works by John Locke, P. Brydone, James Beattie, John Gillies, Plutarch, and Alexander Pope. He describes his work teaching that children of the Sears family in Chatham, Massachusetts, where he appears to have spent a significant amount of time between 1795 and 1797. Church's letters are at times very personal, and he often expresses great affection for Jenks and their friendship.

Notes: concerning sermons : manuscript, 1728-1730

Notes on sermons delivered by John Webb and Peter Thatcher, Congregational ministers at the New North Church in Boston.

Distribution of foraminifera, pre-Pliocene foraminifera and macrofossil debris in sediment core CRP-2 (Table 1)

The stratigraphic distribution, assemblage content, paleoecology and age of foraminifera recovered in fourteen of sixteen samples from the 5.63 m thick CRP-2 (Lithostratigraphic Unit 2.2) are discussed. LSU 2.2 comprises four discrete lithologic beds. The upward sequence is informally referred to as the lower sand bed, diamicton bed, middle sand bed, and upper sand bed and it is surmised that these four units are closely related in time. The lower sand bed (~1.5m), which overlies lower Miocene sediments and from which it is separated by the Ross Sea Unconformity, contains traces of recycled Miocene diatoms but is otherwise barren of biogenic material. The diamicton bed (~2.42 m) contains 21 species of benthic foraminifera, with assemblages consistently dominated by Cassidulinoides porrectus, Ammoelphidiella antarctica, Rosalina cf. globularis, Cibicides refulgens, and Ehrenbergina glabra. The overlying middle sand bed (~1.9 m) contains 13 species. with C. porrectus and E. glabra dominant and A. antarctica less common than in the underlying diamicton bed. The upper sand bed (~0.46 m) contains four species and very few tests. The diamicton bed and middle sand bed assemblages are considered to be near in situ thanatocoenoses; and sediments interpreted as marine in origin but influenced by hyposaline waters and nearby ice. Planktic taxa are absent, perhaps indicating the presence of tidewater glaciers, sea ice and/or hyposaline surface waters. The small assemblage in the upper sand bed is more problematic and may be recycled. On the basis of foraminifera in the diamicton and middle sand beds. LSU 2.2 is assigned to the Pliocene. The overlying diamicton in LSU 2.1 contains abundant Quaternary foraminifera.