p14ARF Post-Transcriptional Regulation of Nuclear Cyclin D1 in MCF-7 Breast Cancer Cells: Discrimination between a Good and Bad Prognosis?

As part of a cell's inherent protection against carcinogenesis, p14ARF is upregulated in response to hyperproliferative signalling to induce cell cycle arrest. This property makes p14ARF a leading candidate for cancer therapy. This study explores the consequences of reactivating p14ARF in breast cancer and the potential of targeting p14ARF in breast cancer treatment. Our results show that activation of the p14ARF-p53-p21-Rb pathway in the estrogen sensitive MCF-7 breast cancer cells induces many hallmarks of senescence including a large flat cell morphology, multinucleation, senescence-associated-β-gal staining, and rapid G1 and G2/M phase cell cycle arrest. P14ARF also induces the expression of the proto-oncogene cyclin D1, which is most often associated with a transition from G1-S phase and is highly expressed in breast cancers with poor clinical prognosis. In this study, siRNA knockdown of cyclin D1, p21 and p53 show p21 plays a pivotal role in the maintenance of high cyclin D1 expression, cell cycle and growth arrest post-p14ARF induction. High p53 and p14ARF expression and low p21/cyclin D1 did not cause cell-cycle arrest. Knockdown of cyclin D1 stops proliferation but does not reverse senescence-associated cell growth. Furthermore, cyclin D1 accumulation in the nucleus post-p14ARF activation correlated with a rapid loss of nucleolar Ki-67 protein and inhibition of DNA synthesis. Latent effects of the p14ARF-induced cellular processes resulting from high nuclear cyclin D1 accumulation included a redistribution of Ki-67 into the nucleoli, aberrant nuclear growth (multinucleation), and cell proliferation. Lastly, downregulation of cyclin D1 through inhibition of ER abrogated latent recurrence. The mediation of these latent effects by continuous expression of p14ARF further suggests a novel mechanism whereby dysregulation of cyclin D1 could have a double-edged effect. Our results suggest that p14ARF induced-senescence is related to late-onset breast cancer in estrogen responsive breast cancers and/or the recurrence of more aggressive breast cancer post-therapy.     

A Short Sequence Motif in the 5′ Leader of the HIV-1 Genome Modulates Extended RNA Dimer Formation and Virus Replication

The 5′ leader of the HIV-1 RNA genome encodes signals that control various steps in the replication cycle, including the dimerization initiation signal (DIS) that triggers RNA dimerization. The DIS folds a hairpin structure with a palindromic sequence in the loop that allows RNA dimerization via intermolecular kissing loop (KL) base pairing. The KL dimer can be stabilized by including the DIS stem nucleotides in the intermolecular base pairing, forming an extended dimer (ED). The role of the ED RNA dimer in HIV-1 replication has hardly been addressed because of technical challenges. We analyzed a set of leader mutants with a stabilized DIS hairpin for in vitro RNA dimerization and virus replication in T cells. In agreement with previous observations, DIS hairpin stability modulated KL and ED dimerization. An unexpected previous finding was that mutation of three nucleotides immediately upstream of the DIS hairpin significantly reduced in vitro ED formation. In this study, we tested such mutants in vivo for the importance of the ED in HIV-1 biology. Mutants with a stabilized DIS hairpin replicated less efficiently than WT HIV-1. This defect was most severe when the upstream sequence motif was altered. Virus evolution experiments with the defective mutants yielded fast replicating HIV-1 variants with second site mutations that (partially) restored the WT hairpin stability. Characterization of the mutant and revertant RNA molecules and the corresponding viruses confirmed the correlation between in vitro ED RNA dimer formation and efficient virus replication, thus indicating that the ED structure is important for HIV-1 replication.     

CRISPRs: Molecular Signatures Used for Pathogen Subtyping

Rapid and accurate strain identification is paramount in the battle against microbial outbreaks, and several subtyping approaches have been developed. One such method uses clustered regular interspaced short palindromic repeats (CRISPRs), DNA repeat elements that are present in approximately half of all bacteria. Though their signature function is as an adaptive immune system against invading DNA such as bacteriophages and plasmids, CRISPRs also provide an excellent framework for pathogen tracking and evolutionary studies. Analysis of the spacer DNA sequences that reside between the repeats has been tremendously useful for bacterial subtyping during molecular epidemiological investigations. Subtyping, or strain identification, using CRISPRs has been employed in diverse Gram-positive and Gram-negative bacteria, including Mycobacterium tuberculosis, Salmonella enterica, and the plant pathogen Erwinia amylovora. This review discusses the several ways in which CRISPR sequences are exploited for subtyping. This includes the well-established spoligotyping methodologies that have been used for 2 decades to type Mycobacterium species, as well as in-depth consideration of newer, higher-throughput CRISPR-based protocols.     

Enumeration of micronucleated CD71-positive human reticulocytes with a single-laser flow cytometer

The extreme rarity of micronucleated reticulocytes (RETs) in the peripheral blood of non-splenectomized humans has precluded facile enumeration of these cells, as well as evaluation of this endpoint as an index of cytogenetic damage. In this report, we describe a high-throughput, single-laser flow cytometric system for scoring the incidence of micronuclei (MN) in newly formed human RETs. The procedure is based on an immunochemical reagent that differentially labels the most immature fraction of RETs from mature erythrocytes based on the expression level of the transferrin receptor (also known as CD71). The resolution of four erythrocyte populations (young RETs and mature erythrocytes, with and without MN) was achieved for human blood cells treated with phycoerythrin-conjugated anti-CD71, RNase, and either SYTOX Green or SYBR Green I nucleic acid dyes. Anti-glycophorin A labeling of erythroid cells (CyChrome conjugate) was also incorporated into the staining procedure to ensure that debris or other potential artifacts did not adversely impact the analyses. Instrument calibration procedures utilizing malaria-infected rodent erythrocytes were also developed, and are described. Using this analytical system, blood samples from 10 healthy non-splenectomized human volunteers were analyzed for micronucleus frequencies with a single-laser flow cytometer. Average micronucleus frequencies in the mature and most immature fraction of RETs were 0.016 and 0.19%, respectively. Blood samples from three healthy splenectomized volunteers were also evaluated. As expected, these samples exhibited higher micronucleus frequencies in the mature subset of erythrocytes (range 0.03-0.18%). The resulting data suggest that MN can be quantified in human erythrocyte populations with a single-laser flow cytometer, and that the frequency of MN cells in the youngest reticulocyte population approaches values expected in the absence of splenic selection against MN-erythrocytes. This high throughput system is potentially important for evaluating the value of the micronucleated reticulocyte endpoint as an index of chromosome breakage and/or chromosome segregational abnormalities in human populations.     

Effects of including an N-terminal insertion region and arginine-mimetic side chains in helical peptoid analogues of lung surfactant protein B

Surfactant protein B (SP-B) is one of two helical, amphipathic proteins critical for the biophysical functioning of lung surfactant (LS) and hence is an important therapeutic protein. This small, complex 79mer has three internal disulfide bonds and homodimerizes via another disulfide bridge. A helical, amphipathic 25mer from the amino terminus (SP-B(1-25)) exhibits surface-active properties similar to those of full-length, synthetic SP-B. In previous work, we created helical, non-natural mimics of SP-B(1-25) based on sequence-specific peptoid 17mers and demonstrated their biomimetic surface activity. Like SP-B(1-25), the peptoids were designed to adopt helical structures with cationic and nonpolar faces. Here, we compare the surface activities of six different helical peptoid analogues of SP-B(1-25) to investigate the importance of mimicking its N-terminal insertion domain as well as its two arginine residues, both thought to be important for the peptide's proper function. Although the peptoid analogues of SP-B(1-25) studied here share many similar features and all functionally mimic SP-B(1-25) to some degree, it is notable that small differences in their sequences and side chain chemistries lead to substantial differences in their observed interactions with a lipid film. A peptoid comprising a hydrophobic, helical insertion region with aromatic side chains shows more biomimetic surface activity than simpler peptoids, and even better activity, by comparison to natural LS, than SP-B(1-25). However, the substitution of lysine-like side chains for arginine-like side chains in the peptoid has little effect on biomimetic surface activity, indicating that interactions of the guanidino groups with lipids may not be critical for the function of these SP-B mimics.     

Endogenous synthesis of corticosteroids in the hippocampus

Background

Brain synthesis of steroids including sex-steroids is attracting much attention. The endogenous synthesis of corticosteroids in the hippocampus, however, has been doubted because of the inability to detect deoxycorticosterone (DOC) synthase, cytochrome P450(c21).

Methodology/Principal Findings

The expression of P450(c21) was demonstrated using mRNA analysis and immmunogold electron microscopic analysis in the adult male rat hippocampus. DOC production from progesterone (PROG) was demonstrated by metabolism analysis of ³H-steroids. All the enzymes required for corticosteroid synthesis including P450(c21), P450(2D4), P450(11β1) and 3β-hydroxysteroid dehydrogenase (3β-HSD) were localized in the hippocampal principal neurons as shown via in situ hybridization and immunoelectron microscopic analysis. Accurate corticosteroid concentrations in rat hippocampus were determined by liquid chromatography-tandem mass spectrometry. In adrenalectomized rats, net hippocampus-synthesized corticosterone (CORT) and DOC were determined to 6.9 and 5.8 nM, respectively. Enhanced spinogenesis was observed in the hippocampus following application of low nanomolar (10 nM) doses of CORT for 1 h.

Conclusions/Significance

These results imply the complete pathway of corticosteroid synthesis of ‘pregnenolone →PROG→DOC→CORT’ in the hippocampal neurons. Both P450(c21) and P450(2D4) can catalyze conversion of PROG to DOC. The low nanomolar level of CORT synthesized in hippocampal neurons may play a role in modulation of synaptic plasticity, in contrast to the stress effects by micromolar CORT from adrenal glands.     

A fluorescence polarization assay using an engineered human respiratory syncytial virus F protein as a direct screening platform

Probe electrospray ionization (PESI) is one of the most promising methods in biochemical analysis because it enables us to analyze biological samples very quickly without any special pretreatment. Moreover, due to the small size of the needle tip, this method has advantages such as low invasiveness to the samples, making it possible to analyze the biological profiles of organs or tissues in living animal in situ. In this study, we performed a real-time analysis of living mice that delineates the differences in lipid composition of hepatocytes between normal and steatotic mice. In steatotic mice, the number of peaks and the ion abundance for triacylglycerols were much higher compared with those of control mice. All mice used in this study tolerated the procedure well and survived for more than a month until sacrificed for further analysis. To test a potential for medical diagnosis, human tumor tissues were also measured and we obtained discriminative results judged as useful for diagnostics. These results pave the way into the application of PESI to the in vivo analysis of biological molecules.     

Selective modulation of task performance by octopamine in honey bee (<Emphasis Type="Italic">Apis mellifera</Emphasis>) division of labour

Octopamine treatment has previously been shown to increase honey bee foraging behaviour. We determined the effects of octopamine on other tasks to learn how octopamine affects division of labour in honey bee colonies. Octopamine treatment did not increase the rate of corpse removal from the hive, suggesting that elevated brain levels of octopamine do not act to increase the performance of all flight-related tasks. Octopamine treatment also did not increase attendance in the queen's retinue, suggesting that elevated brain levels of octopamine do not act to increase responsiveness to all olfactory stimuli. Consistent with these findings, octopamine treatment enhanced the foraging response to brood pheromone but not the cell capping response, a component of brood care. These results demonstrate a relatively specific form of neuromodulation by octopamine in the regulation of division of labour in honey bee colonies.