siRNA-Mediated Knock-Down of P-Glycoprotein Expression Reveals Distinct Cellular Disposition of Anticancer Tyrosine Kinases Inhibitors.

Studies on the cellular disposition of targeted anticancer tyrosine kinases inhibitors (TKIs) have mostly focused on imatinib while the functional importance of P-glycoprotein (Pgp) the gene product of MDR1 remains controversial for more recent TKIs. By using RNA interference-mediated knockdown of MDR1, we have investigated and compared the specific functional consequence of Pgp on the cellular disposition of the major clinically in use TKIs imatinib, dasatinib, nilotinib, sunitinib and sorafenib. siRNA-mediated knockdown in K562/Dox cell lines provides a unique opportunity to dissect the specific contribution of Pgp to TKIs intracellular disposition. In these conditions, abrogating specifically Pgp-mediated efflux in vitro revealed the remarkable and statistically significant cellular accumulation of imatinib (difference in cellular levels between Pgp-expressing and silenced cells, at high and low incubation concentration, respectively: 6.1 and 6.6), dasatinib (4.9 and 5.6), sunitinib (3.7 and 7.3) and sorafenib (1.2 and 1.4), confirming that these TKIs are all substrates of Pgp. By contrast, no statistically significant difference in cellular disposition of nilotinib was observed as a result of MDR1 expression silencing (differences: 1.1 and 1.5) indicating that differential expression and/or function of Pgp is unlikely to affect nilotinib cellular disposition. This study enables for the first time a direct estimation of the specific contribution of one transporter among the various efflux and influx carriers involved in the cellular trafficking of these major TKIs in vitro. Knowledge on the distinct functional consequence of Pgp expression for these various TKIs cellular distribution is necessary to better appreciate the efficacy, toxicity, and potential drug-drug interactions of TKIs with other classes of therapeutic agents, at the systemic, tissular and cellular levels.

A single strain of Mycobacterium bovis bacillus Calmette-Guérin (BCG) grown in two different media evokes distinct humoral immune responses in mice

Two attenuated bacillus Calmette-Guérin (BCG) preparations derived from the same Moreau strain, Copenhagen but grown in Sauton medium containing starch and bacto-peptone (onco BCG, O-BCG), or asparagine (intradermal BCG, ID-BCG), exhibited indistinguishable DNA sequences and bacterial morphology. The number of viable bacilli recovered from spleen, liver and lungs was approximately the same in mice inoculated with the vaccines and was similarly reduced (over 90%) in mice previously immunized with either BCG vaccine. The humoral immune response evoked by the vaccines was, however, distinct. Spleen cell proliferation accompanying the growth of bacilli in tissue was significantly higher in mice inoculated with O-BCG. These cells proliferated in vitro upon challenge with the corresponding BCG extract. Previous cell treatment with mAb anti-CD4 T cells abolished this effect. Anti-BCG antibodies, as assayed either in serum by ELISA or by determining the number of antibody-producing spleen cells by the spot-ELISA method, were significantly higher in mice inoculated with ID-BCG. Anti-BCG antibodies were detected in all immunoglobulin classes, but they were more prevalent in IgG with the following distribution among its isotypes: IgG1>(IgG2a = IgG2b)>IgG3. When some well-characterized Mycobacterium tuberculosis antigens were used as substitutes for BCG extracts in ELISA, although antibodies against the 65-kDa and 96-kDa proteins were detected significantly, antibodies against the 71-kDa, 38-kDa proteins and lipoarabinomannan were only barely detected or even absent. These results indicate that BCG bacilli cultured in Sauton-asparagine medium permitted the multiplication of bacilli, tending to induce a stronger humoral immune response as compared with bacilli grown in Sauton-starch/bacto-peptone-enriched medium.

Radial frequency stimuli and sine-wave gratings seem to be processed by distinct contrast brain mechanisms

An assumption commonly made in the study of visual perception is that the lower the contrast threshold for a given stimulus, the more sensitive and selective will be the mechanism that processes it. On the basis of this consideration, we investigated contrast thresholds for two classes of stimuli: sine-wave gratings and radial frequency stimuli (i.e., j0 targets or stimuli modulated by spherical Bessel functions). Employing a suprathreshold summation method, we measured the selectivity of spatial and radial frequency filters using either sine-wave gratings or j0 target contrast profiles at either 1 or 4 cycles per degree of visual angle (cpd), as the test frequencies. Thus, in a forced-choice trial, observers chose between a background spatial (or radial) frequency alone and the given background stimulus plus the test frequency (1 or 4 cpd sine-wave grating or radial frequency). Contrary to our expectations, the results showed elevated thresholds (i.e., inhibition) for sine-wave gratings and decreased thresholds (i.e., summation) for radial frequencies when background and test frequencies were identical. This was true for both 1- and 4-cpd test frequencies. This finding suggests that sine-wave gratings and radial frequency stimuli are processed by different quasi-linear systems, one working at low luminance and contrast level (sine-wave gratings) and the other at high luminance and contrast levels (radial frequency stimuli). We think that this interpretation is consistent with distinct foveal only and foveal-parafoveal mechanisms involving striate and/or other higher visual areas (i.e., V2 and V4).

On The Center Sets and Center Numbers of Some Graph Classes

For a set S of vertices and the vertex v in a connected graph G, max x2S d(x, v) is called the S-eccentricity of v in G. The set of vertices with minimum S-eccentricity is called the S-center of G. Any set A of vertices of G such that A is an S-center for some set S of vertices of G is called a center set. We identify the center sets of certain classes of graphs namely, Block graphs, Km,n, Kn −e, wheel graphs, odd cycles and symmetric even graphs and enumerate them for many of these graph classes. We also introduce the concept of center number which is defined as the number of distinct center sets of a graph and determine the center number of some graph classes

The global distribution of phytoplankton size spectrum and size classes from their light-absorption spectra derived from satellite data

The absorption spectra of phytoplankton in the visible domain hold implicit information on the phytoplankton community structure. Here we use this information to retrieve quantitative information on phytoplankton size structure by developing a novel method to compute the exponent of an assumed power-law for their particle-size spectrum. This quantity, in combination with total chlorophyll-a concentration, can be used to estimate the fractional concentration of chlorophyll in any arbitrarily-defined size class of phytoplankton. We further define and derive expressions for two distinct measures of cell size of mixed populations, namely, the average spherical diameter of a bio-optically equivalent homogeneous population of cells of equal size, and the average equivalent spherical diameter of a population of cells that follow a power-law particle-size distribution. The method relies on measurements of two quantities of a phytoplankton sample: the concentration of chlorophyll-a, which is an operational index of phytoplankton biomass, and the total absorption coefficient of phytoplankton in the red peak of visible spectrum at 676 nm. A sensitivity analysis confirms that the relative errors in the estimates of the exponent of particle size spectra are reasonably low. The exponents of phytoplankton size spectra, estimated for a large set of in situ data from a variety of oceanic environments (~ 2400 samples), are within a reasonable range; and the estimated fractions of chlorophyll in pico-, nano- and micro-phytoplankton are generally consistent with those obtained by an independent, indirect method based on diagnostic pigments determined using high-performance liquid chromatography. The estimates of cell size for in situ samples dominated by different phytoplankton types (diatoms, prymnesiophytes, Prochlorococcus, other cyanobacteria and green algae) yield nominal sizes consistent with the taxonomic classification. To estimate the same quantities from satellite-derived ocean-colour data, we combine our method with algorithms for obtaining inherent optical properties from remote sensing. The spatial distribution of the size-spectrum exponent and the chlorophyll fractions of pico-, nano- and micro-phytoplankton estimated from satellite remote sensing are in agreement with the current understanding of the biogeography of phytoplankton functional types in the global oceans. This study contributes to our understanding of the distribution and time evolution of phytoplankton size structure in the global oceans.

Chromosomal Variability among Allopatric Populations of Erythrinidae Fish Hoplias malabaricus: Mapping of Three Classes of Repetitive DNAs

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Documenting the advantages and limitations of different classes of molecular markers in a well-established phylogeographic context: lessons from the Iberian endemic Golden-striped salamander, Chioglossa lusitanica (Caudata: Salamandridae)

Previous analyses of mitochondrial (mt)DNA and allozymes covering the range of the Iberian endemic golden-striped salamander, Chioglossa lusitanica, suggested a Pleistocene split of the historical species distribution into two population units (north and south of the Mondego river), postglacial expansion into the northernmost extant range, and secondary contact with neutral diffusion of genes close to the Mondego river. We extended analysis of molecular variation over the species range using seven microsatellite loci and the nuclear P-fibrinogen intron 7 (beta-fibint7). Both microsatellites and beta-fibint7 showed moderate to high levels of population structure, concordant with patterns detected with mtDNA and allozymes; and a general pattern of isolation-by-distance, contrasting the marked differentiation of two population groups suggested by mtDNA and allozymes. Bayesian multilocus analyses showed contrasting results as populations north and south of the Douro river were clearly differentiated based on microsatellites, whereas allozymes revealed differentiation north and south of the Mondego river. Additionally, decreased microsatellite variability in the north supported the hypothesis of postglacial colonization of this region. The well-documented evolutionary history of C. lusitanica, provides an excellent framework within which the advantages and limitations of different classes of markers can be evaluated in defining patterns of population substructure and inferring evolutionary processes across distinct spatio-temporal scales. The present study serves as a cautionary note for investigations that rely on a single type of molecular marker, especially when the organism under study exhibits a widespread distribution and complex natural history. (C) 2008 The Linnean Society of London, Biological Journal of the Linnean Society, 2008, 95, 371-387.

Drosophila host defense: Differential induction of antimicrobial peptide genes after infection by various classes of microorganisms

Insects respond to microbial infection by the rapid and transient expression of several genes encoding potent antimicrobial peptides. Herein we demonstrate that this antimicrobial response of Drosophila is not aspecific but can discriminate between various classes of microorganisms. We first observe that the genes encoding antibacterial and antifungal peptides are differentially expressed after injection of distinct microorganisms. More strikingly, Drosophila that are naturally infected by entomopathogenic fungi exhibit an adapted response by producing only peptides with antifungal activities. This response is mediated through the selective activation of the Toll pathway.

Stimulation of bacteriophage T4 middle transcription by the T4 proteins MotA and AsiA occurs at two distinct steps in the transcription cycle

The bacteriophage T4 encodes proteins that are responsible for tightly regulating mRNA synthesis throughout phage development in Escherichia coli. The three classes of T4 promoters (early, middle, and late) are utilized sequentially by the host RNA polymerase as a result of phage-induced modifications. One such modification is the tight binding of the T4 AsiA protein to the σ70 subunit of the RNA polymerase. This interaction is pivotal for the transition between T4 early and middle transcription, since it both inhibits recognition of host and T4 early promoters and stimulates T4 middle mode synthesis. The activation of T4 middle transcription also requires the T4 MotA protein, bound specifically to its recognition sequence, the “Mot box,” which is centered at position −30 of these promoters. Accordingly, the two T4 proteins working in concert are sufficient to effectively switch the transcription specificity of the RNA polymerase holoenzyme. Herein, we investigate the mechanism of transcription activation and report that, while the presence of MotA and AsiA increases the initial recruitment of RNA polymerase to a T4 middle promoter, it does not alter the intrinsic stability of the discrete complexes formed. In addition, we have characterized the RNA polymerase-promoter species by UV laser footprinting and followed their evolution from open into initiating complexes. These data, combined with in vitro transcription assays, indicate that AsiA and MotA facilitate promoter escape, thereby stimulating the production of full-length transcripts.

Distinct Modes of Macrophage Recognition for Apoptotic and Necrotic Cells Are Not Specified Exclusively by Phosphatidylserine Exposure

The distinction between physiological (apoptotic) and pathological (necrotic) cell deaths reflects mechanistic differences in cellular disintegration and is of functional significance with respect to the outcomes that are triggered by the cell corpses. Mechanistically, apoptotic cells die via an active and ordered pathway; necrotic deaths, conversely, are chaotic and passive. Macrophages and other phagocytic cells recognize and engulf these dead cells. This clearance is believed to reveal an innate immunity, associated with inflammation in cases of pathological but not physiological cell deaths. Using objective and quantitative measures to assess these processes, we find that macrophages bind and engulf native apoptotic and necrotic cells to similar extents and with similar kinetics. However, recognition of these two classes of dying cells occurs via distinct and noncompeting mechanisms. Phosphatidylserine, which is externalized on both apoptotic and necrotic cells, is not a specific ligand for the recognition of either one. The distinct modes of recognition for these different corpses are linked to opposing responses from engulfing macrophages. Necrotic cells, when recognized, enhance proinflammatory responses of activated macrophages, although they are not sufficient to trigger macrophage activation. In marked contrast, apoptotic cells profoundly inhibit phlogistic macrophage responses; this represents a cell-associated, dominant-acting anti-inflammatory signaling activity acquired posttranslationally during the process of physiological cell death.

Precise sequence complementarity between yeast chromosome ends and two classes of just-subtelomeric sequences

The terminal regions (last 20 kb) of Saccharomyces cerevisiae chromosomes universally contain blocks of precise sequence similarity to other chromosome terminal regions. The left and right terminal regions are distinct in the sense that the sequence similarities between them are reverse complements. Direct sequence similarity occurs between the left terminal regions and also between the right terminal regions, but not between any left ends and right ends. With minor exceptions the relationships range from 80% to 100% match within blocks. The regions of similarity are composites of familiar and unfamiliar repeated sequences as well as what could be considered “single-copy” (or better “two-copy”) sequences. All terminal regions were compared with all other chromosomes, forward and reverse complement, and 768 comparisons are diagrammed. It appears there has been an extensive history of sequence exchange or copying between terminal regions. The subtelomeric sequences fall into two classes. Seventeen of the chromosome ends terminate with the Y′ repeat, while 15 end with the 800-nt “X2” repeats just adjacent to the telomerase simple repeats. The just-subterminal repeats are very similar to each other except that chromosome 1 right end is more divergent.

Use of genetic suppressor elements to dissect distinct biological effects of separate p53 domains.

p53 is a multifunctional tumor suppressor protein involved in the negative control of cell growth. Mutations in p53 cause alterations in cellular phenotype, including immortalization, neoplastic transformation, and resistance to DNA-damaging drugs. To help dissect distinct functions of p53, a set of genetic suppressor elements (GSEs) capable of inducing different p53-related phenotypes in rodent embryo fibroblasts was isolated from a retroviral library of random rat p53 cDNA fragments. All the GSEs were 100-300 nucleotides long and were in the sense orientation. They fell into four classes, corresponding to the transactivator (class I), DNA-binding (class II), and C-terminal (class III) domains of the protein and the 3'-untranslated region of the mRNA (class IV). GSEs in all four classes promoted immortalization of primary cells, but only members of classes I and III cooperated with activated ras to transform cells, and only members of class III conferred resistance to etoposide and strongly inhibited transcriptional transactivation by p53. These observations suggest that processes related to control of senescence, response to DNA damage, and transformation involve different functions of the p53 protein and furthermore indicate a regulatory role for the 3'-untranslated region of p53 mRNA.

Three distinct structural environments of a transmembrane domain in the inwardly rectifying potassium channel ROMK1 defined by perturbation.

To probe the protein environment of an ion channel, we have perturbed the structure of a transmembrane domain by substituting side chains with those of two different sizes by using site-specific mutagenesis. We have used Trp and Ala as a high- and a low-impact perturbation probe, respectively, to replace each of 18 consecutive residues within the putative second transmembrane segment, M2, of an inwardly rectifying potassium channel, ROMK1. Our rationale is that a change in the channel function as a consequence of these mutations at a particular position will reflect the structural environment of the altered side chain. Each position can then be assigned to one of three classes of environments, as grated by different levels of perturbation: very tolerant (channel functions with both Trp and Ala substitutions), tolerant (function preserved with Ala but not with Trp substitution), and intolerant (either Ala or Trp substitution destroys function). We identify the very tolerant environment as being lipid-facing, tolerant as protein-interior-facing, and intolerant as pore-facing. We observe a strikingly ordered pattern of perturbation of all three environmental classes. This result indicates that M2 is a straight alpha-helix.

Cloning of a receptor for amphibian [Phe13]bombesin distinct from the receptor for gastrin-releasing peptide: identification of a fourth bombesin receptor subtype (BB4).

Bombesin is a tetradecapeptide originally isolated from frog skin and demonstrated to have a wide range of actions in mammals. Based on structural homology and similar biological activities, gastrin-releasing peptide (GRP) has been considered the mammalian equivalent of bombesin. We previously reported that frogs have both GRP and bombesin, which therefore are distinct peptides. We now report the cloning of a bombesin receptor subtype (BB4) that has higher affinity for bombesin than GRP. PCR was used to amplify cDNAs related to the known bombesin receptors from frog brain. Sequence analysis of the amplified cDNAs revealed 3 classes of receptor subtypes. Based on amino acid homology, two classes were clearly the amphibian homologs of the GRP and neuromedin B receptors. The third class was unusual and a full-length clone was isolated from a Bombina orientalis brain cDNA library. Expression of the receptor in Xenopus oocytes demonstrated that the receptor responded to picomolar concentrations of [Phe13]-bombesin, the form of bombesin most prevalent in frog brain. The relative rank potency of bombesin-like peptides for this receptor was [Phe13]bombesin > [Leu13]bombesin > GRP > neuromedin B. In contrast, the rank potency for the GRP receptor is GRP > [Leu13]bombesin > [Phe13]bombesin > neuromedin B. Transient expression in CHOP cells gave a Ki for [Phe13]bombesin of 0.2 nM versus a Ki of 2.1 nM for GRP. Distribution analysis showed that this receptor was expressed only in brain, consistent with the distribution of [Phe13]-bombesin. Thus, based on distribution and affinity, this bombesin receptor is the receptor for [Phe13]bombesin. Phylogenetic analysis suggests that this receptor separated prior to separation of the GRP and neuromedin B receptors; thus, BB4 receptors and their cognate ligands may also exist in mammals.

Migraine With Aura and Migraine Without Aura Are Not Distinct Entities: Further Evidence From a Large Dutch Population Study

It is often debated whether migraine with aura (MA) and migraine without aura (MO) are etiologically distinct disorders. A previous study using latent class analysis (LCA) in Australian twins showed no evidence for separate subtypes of MO and MA. The aim of the present study was to replicate these results in a population of Dutch twins and their parents, siblings and partners (N = 10,144). Latent class analysis of International Headache Society (IHS)-based migraine symptoms resulted in the identification of 4 classes: a class of unaffected subjects (class 0), a mild form of nonmigrainous headache (class 1), a moderately severe type of migraine (class 2), typically without neurological symptoms or aura (8% reporting aura symptoms), and a severe type of migraine (class 3), typically with neurological symptoms, and aura symptoms in approximately half of the cases. Given the overlap of neurological symptoms and nonmutual exclusivity of aura symptoms, these results do not support the MO and MA subtypes as being etiologically distinct. The heritability in female twins of migraine based on LCA classification was estimated at .50 (95% confidence intervals [0CI} .27 -.59), similar to IHS-based migraine diagnosis (h(2) = .49, 95% Cl .19-.57). However, using a dichotomous classification (affected-unaffected) decreased heritability for the IHS-based classification (h(2) = .33, 95% Cl .00-.60), but not the LCA-based classification (h(2) = .51, 95% Cl. 23-.61). Importantly, use of the LCA-based classification increased the number of subjects classified as affected. The heritability of the screening question was similar to more detailed LCA and IHS classifications, suggesting that the screening procedure is an important determining factor in genetic studies of migraine.