Dimethylsphingosine evokes diverse cellular death modes in HL60

Among the barriers to successful cancer treatment is the acquired resistance that tumors undergo due to clonal evolution. Non-cross resistant drugs could add to the current options of chemotherapeutic drugs. In order to improve tumor response, investigators have been identifying defective death pathways acquired by specific cancer types, so to target directly those pathways. Sphingolipids have emerged as potential drugs for tumor-targeted therapy, and among them, Dimethylsphingosine (DMS), known to be a competitive inhibitor of Sphingosine Kinase (SK). DMS actions have been documented by several investigators, but the mechanisms by which DMS exerts cytotoxicity have not been fully investigated. We evaluated the cytotoxicity of DMS against human leukemia cell lines and against blasts isolated from leukemia patients. Cell line viability decreased proportionally to DMS concentration and treatment time. Resistant and MDR positive cell lines were the most sensitive, indicating DMS efficacy against human leukemia MDR. Importantly, leukemia samples showed a similar sensitivity to DMS, the first demonstration of DMS activity against fresh human leukemia specimens. Mechanistically we have demonstrated that DMS efficacy is due to its ability to induce cytotoxicity by inducing necrosis, apoptosis or both concomitantly, revealing a mixed-feature cell death mode never described before for DMS. Further, we have shown evidence suggesting pathways cross-talk, since apoptosis inhibition led to accelerated rate of necrosis. DMS diverse killing mechanisms and the high expression of SK in leukemias could explain DMS potent cytotoxicity. DMS-based regimens may increase response rates and therefore, improve leukemia treatment. ^

Preservice Teacher Preparation in Meeting the Needs of Gifted and Other Academically Diverse Students

The issue of how to respond to the diverse academic needs of students is one of the central challenges of teaching. This project studied how preservice teachers develop an awareness of the needs of academically diverse learners and how they intend to implement and/or modify instruction to meet those needs. Participants all came from one university. As part of the design of the study, the participants were surveyed to investigate (a) their attitudes and beliefs towards academically diverse learners; (b) the teaching practices they would utilize in response to academic diversity in their classrooms; and (c) the confidence they have in their abilities to identify and address these various needs in their classrooms. Several strategies including activities to enhance creativity, cooperative learning, individual instruction, problem-solving activities, and projects were considered noteworthy for the ratings by the preservice teachers as appropriate for all students. Small differences were found based on the preservice teachers' year of placement in the School of Education, indicating that as students progress through this program, they may learn more about different techniques and when and for whom they are appropriate; however, differences across groups were not statistically significant. Results also indicated that across the different years in the program, preservice teachers did not have very high or very low confidence in addressing these issues in their own classrooms. Each grouping fell around the middle level of confidence.

Diverse coral communities in naturally acidified waters of a Western Pacific reef

Anthropogenic carbon dioxide emissions are acidifying the oceans, reducing the concentration of carbonate ions ([CO32-) that calcifying organisms need to build and cement coral reefs. To date, studies of a handful of naturally acidified reef systems reveal depauperate communities, sometimes with reduced coral cover and calcification rates, consistent with results of laboratory-based studies. Here we report the existence of highly diverse, coral-dominated reef communities under chronically low pH and aragonite saturation state (Omega ar). Biological and hydrographic processes change the chemistry of the seawater moving across the barrier reefs and into Palau's Rock Island bays, where levels of acidification approach those projected for the western tropical Pacific open ocean by 2100. Nevertheless, coral diversity, cover, and calcification rates are maintained across this natural acidification gradient. Identifying the combination of biological and environmental factors that enable these communities to persist could provide important insights into the future of coral reefs under anthropogenic acidification.

Raccolta di diverse invenzioni di nº 24 Fabbriche : Contenenti chiese ospedali palazzi casini di campagna ed altre: Incise a bulino in nº 24 tavole con le loro respettive piante e spaccati

Port. con grab. calc

Ectopic expression of the minimal whiE polyketide synthase generates a library of aromatic polyketides of diverse sizes and shapes

The single recombinant expressing the Streptomyces coelicolor minimal whiE (spore pigment) polyketide synthase (PKS) is uniquely capable of generating a large array of well more than 30 polyketides, many of which, so far, are novel to this recombinant. The characterized polyketides represent a diverse set of molecules that differ in size (chain length) and shape (cyclization pattern). This combinatorial biosynthetic library is, by far, the largest and most complex of its kind described to date and indicates that the minimal whiE PKS does not independently control polyketide chain length nor dictate the first cyclization event. Rather, the minimal PKS enzyme complex must rely on the stabilizing effects of additional subunits (i.e., the cyclase whiE-ORFVI) to ensure that the chain reaches the full 24 carbons and cyclizes correctly. This dramatic loss of control implies that the growing polyketide chain does not remain enzyme bound, resulting in the spontaneous cyclization of the methyl terminus. Among the six characterized dodecaketides, four different first-ring cyclization regiochemistries are represented, including C7/C12, C8/C13, C10/C15, and C13/C15. The dodecaketide TW93h possesses a unique 2,4-dioxaadamantane ring system and represents a new structural class of polyketides with no related structures isolated from natural or engineered organisms, thus supporting the claim that engineered biosynthesis is capable of producing novel chemotypes.

Sequence-specific polypeptoids: A diverse family of heteropolymers with stable secondary structure

We have synthesized and characterized a family of structured oligo-N-substituted-glycines (peptoids) up to 36 residues in length by using an efficient solid-phase protocol to incorporate chemically diverse side chains in a sequence-specific fashion. We investigated polypeptoids containing side chains with a chiral center adjacent to the main chain nitrogen. Some of these sequences have stable secondary structure, despite the achirality of the polymer backbone and its lack of hydrogen bond donors. In both aqueous and organic solvents, peptoid oligomers as short as five residues give rise to CD spectra that strongly resemble those of peptide α-helices. Differential scanning calorimetry and CD measurements show that polypeptoid secondary structure is highly stable and that unfolding is reversible and cooperative. Thermodynamic parameters obtained for unfolding are similar to those obtained for the α-helix to coil transitions of peptides. This class of biomimetic polymers may enable the design of self-assembling macromolecules with novel structures and functions.

Nucleus reticularis neurons mediate diverse inhibitory effects in thalamus

Detailed information regarding the contribution of individual γ-aminobutyric acid (GABA)-containing inhibitory neurons to the overall synaptic activity of single postsynaptic cells is essential to our understanding of fundamental elements of synaptic integration and operation of neuronal circuits. For example, GABA-containing cells in the thalamic reticular nucleus (nRt) provide major inhibitory innervation of thalamic relay nuclei that is critical to thalamocortical rhythm generation. To investigate the contribution of individual nRt neurons to the strength of this internuclear inhibition, we obtained whole-cell recordings of unitary inhibitory postsynaptic currents (IPSCs) evoked in ventrobasal thalamocortical (VB) neurons by stimulation of single nRt cells in rat thalamic slices, in conjunction with intracellular biocytin labeling. Two types of monosynaptic IPSCs could be distinguished. “Weak” inhibitory connections were characterized by a significant number of postsynaptic failures in response to presynaptic nRt action potentials and relatively small IPSCs. In contrast, “strong” inhibition was characterized by the absence of postsynaptic failures and significantly larger unitary IPSCs. By using miniature IPSC amplitudes to infer quantal size, we estimated that unitary IPSCs associated with weak inhibition resulted from activation of 1–3 release sites, whereas stronger inhibition would require simultaneous activation of 5–70 release sites. The inhibitory strengths were positively correlated with the density of axonal swellings of the presynaptic nRt neurons, an indicator that characterizes different nRt axonal arborization patterns. These results demonstrate that there is a heterogeneity of inhibitory interactions between nRt and VB neurons, and that variations in gross morphological features of axonal arbors in the central nervous system can be associated with significant differences in postsynaptic response characteristics.

A mitogen-activated protein kinase of the corn leaf pathogen Cochliobolus heterostrophus is involved in conidiation, appressorium formation, and pathogenicity: Diverse roles for mitogen-activated protein kinase homologs in foliar pathogens

Fungal pathogens perceive and respond to molecules from the plant, triggering pathogenic development. Transduction of these signals may use heterotrimeric G proteins, and it is thought that protein phosphorylation cascades are also important. We have isolated a mitogen-activated protein kinase homolog from the corn pathogen Cochliobolus heterostrophus to test its role as a component of the transduction pathways. The new gene, CHK1, has a deduced amino acid sequence 90% identical to Pmk1 of the rice blast fungus Magnaporthe grisea and 59% identical to Fus3 of Saccharomyces cerevisiae. A series of chk1 deletion mutants has poorly developed aerial hyphae, autolysis, and no conidia. No pseudothecia are formed when a cross between two Δchk1 mutants is attempted. The ability of Δchk1 mutants to infect corn plants is reduced severely. The growth pattern of hyphae on a glass surface is strikingly altered from that of the wild type, forming coils or loops, but no appressoria. This set of phenotypes overlaps only partially with that of pmk1 mutants, the homologous gene of the rice blast fungus. In particular, sexual and asexual sporulation both require Chk1 function in Cochliobolus heterostrophus, in contrast to Pmk1, but perhaps more similar to yeast, where Fus3 transmits the mating signal. Chk1 is required for efficient colonization of leaf tissue, which can be compared with filamentous invasive growth of yeast, modulated through another closely related mitogen-activated protein kinase, Kss1. Ubiquitous signaling elements thus are used in diverse ways in different plant pathogens, perhaps the result of coevolution of the transducers and their targets.

In silico detection of control signals: mRNA 3′-end-processing sequences in diverse species

We have investigated mRNA 3′-end-processing signals in each of six eukaryotic species (yeast, rice, arabidopsis, fruitfly, mouse, and human) through the analysis of more than 20,000 3′-expressed sequence tags. The use and conservation of the canonical AAUAAA element vary widely among the six species and are especially weak in plants and yeast. Even in the animal species, the AAUAAA signal does not appear to be as universal as indicated by previous studies. The abundance of single-base variants of AAUAAA correlates with their measured processing efficiencies. As found previously, the plant polyadenylation signals are more similar to those of yeast than to those of animals, with both common content and arrangement of the signal elements. In all species examined, the complete polyadenylation signal appears to consist of an aggregate of multiple elements. In light of these and previous results, we present a broadened concept of 3′-end-processing signals in which no single exact sequence element is universally required for processing. Rather, the total efficiency is a function of all elements and, importantly, an inefficient word in one element can be compensated for by strong words in other elements. These complex patterns indicate that effective tools to identify 3′-end-processing signals will require more than consensus sequence identification.

Suppression of gene silencing: A general strategy used by diverse DNA and RNA viruses of plants

In transgenic and nontransgenic plants, viruses are both initiators and targets of a defense mechanism that is similar to posttranscriptional gene silencing (PTGS). Recently, it was found that potyviruses and cucumoviruses encode pathogenicity determinants that suppress this defense mechanism. Here, we test diverse virus types for the ability to suppress PTGS. Nicotiana benthamiana exhibiting PTGS of a green fluorescent protein transgene were infected with a range of unrelated viruses and various potato virus X vectors producing viral pathogenicity factors. Upon infection, suppression of PTGS was assessed in planta through reactivation of green fluorescence and confirmed by molecular analysis. These experiments led to the identification of three suppressors of PTGS and showed that suppression of PTGS is widely used as a counter-defense strategy by DNA and RNA viruses. However, the spatial pattern and degree of suppression varied extensively between viruses. At one extreme, there are viruses that suppress in all tissues of all infected leaves, whereas others are able to suppress only in the veins of new emerging leaves. This variation existed even between closely related members of the potexvirus group. Collectively, these results suggest that virus-encoded suppressors of gene silencing have distinct modes of action, are targeted against distinct components of the host gene-silencing machinery, and that there is dynamic evolution of the host and viral components associated with the gene-silencing mechanism.

SET1, A Yeast Member of the Trithorax Family, Functions in Transcriptional Silencing and Diverse Cellular Processes

The trithorax gene family contains members implicated in the control of transcription, development, chromosome structure, and human leukemia. A feature shared by some family members, and by other proteins that function in chromatin-mediated transcriptional regulation, is the presence of a 130- to 140-amino acid motif dubbed the SET or Tromo domain. Here we present analysis of SET1, a yeast member of the trithorax gene family that was identified by sequence inspection to encode a 1080-amino acid protein with a C-terminal SET domain. In addition to its SET domain, which is 40–50% identical to those previously characterized, SET1 also shares dispersed but significant similarity to Drosophila and human trithorax homologues. To understand SET1 function(s), we created a null mutant. Mutant strains, although viable, are defective in transcriptional silencing of the silent mating-type loci and telomeres. The telomeric silencing defect is rescued not only by full-length episomal SET1 but also by the conserved SET domain of SET1. set1 mutant strains display other phenotypes including morphological abnormalities, stationary phase defects, and growth and sporulation defects. Candidate genes that may interact with SET1 include those with functions in transcription, growth, and cell cycle control. These data suggest that yeast SET1, like its SET domain counterparts in other organisms, functions in diverse biological processes including transcription and chromatin structure.

Conserved circadian elements in phylogenetically diverse algae

Circadian expression of the luciferin-binding protein (LBP) from the dinoflagellate Gonyaulax polyedra is regulated at the translational level. A small interval in the lbp 3′-untranslated region, which contains seven UG-repeats, serves as a cis-acting element to which a trans-acting factor (CCTR) binds in a circadian manner. Its binding activity correlates negatively with the circadian expression of LBP. Here I report the identification of a protein in the green alga Chlamydomonas reinhardtii that represents a CCTR analog. It binds both specifically and under control of the circadian clock to the UG-repeat region. The data show for the first time that circadian cis-elements implicated in translational regulation have been conserved during evolution.