StearoylCoA desaturase-5: a novel regulator of neuronal cell proliferation and differentiation

Recent studies have demonstrated that human stearoylCoA desaturase-1 (SCD1), a Δ9-desaturase that converts saturated fatty acids (SFA) into monounsaturated fatty acids, controls the rate of lipogenesis, cell proliferation and tumorigenic capacity in cancer cells. However, the biological function of stearoylCoA desaturase-5 (SCD5), a second isoform of human SCD that is highly expressed in brain, as well as its potential role in human disease, remains unknown. In this study we report that the constitutive overexpression of human SCD5 in mouse Neuro2a cells, a widely used cell model of neuronal growth and differentiation, displayed a greater n-7 MUFA-to-SFA ratio in cell lipids compared to empty-vector transfected cells (controls). De novo synthesis of phosphatidylcholine and cholesterolesters was increased whereas phosphatidylethanolamine and triacylglycerol formation was reduced in SCD5-expressing cells with respect to their controls, suggesting a differential use of SCD5 products for lipogenic reactions. We also observed that SCD5 expression markedly accelerated the rate of cell proliferation and suppressed the induction of neurite outgrowth, a typical marker of neuronal differentiation, by retinoic acid indicating that the desaturase plays a key role in the mechanisms of cell division and differentiation. Critical signal transduction pathways that are known to modulate these processes, such epidermal growth factor receptor (EGFR)Akt/ERK and Wnt, were affected by SCD5 expression. Epidermal growth factor-induced phosphorylation of EGFR, Akt and ERK was markedly blunted in SCD5-expressing cells. Furthermore, the activity of canonical Wnt was reduced whereas the non-canonical Wnt was increased by the presence of SCD5 activity. Finally, SCD5 expression increased the secretion of recombinant Wnt5a, a non-canonical Wnt, whereas it reduced the cellular and secreted levels of canonical Wnt7b. Our data suggest that, by a coordinated modulation of key lipogenic pathways and transduction signaling cascades, SCD5 participates in the regulation of neuronal cell growth and differentiation.     

Effect of suppression of erythropoiesis on hematopoietic stem cells in the mouse

Erythropoiesis was suppressed in CDF₁ female mice using one of three different techniques: hypertransfusion, daily injections of low doses of actinomycin D, or post-hypobaric polycythemia. Spleens and bone marrows of these mice contained increased numbers of stem cells as determined by transplantation into lethally irradiated syngeneic mice. These stem cells were capable of producing both erythrocytic and granulocytic progeny. An increase in the number of multipotential stem cells or increases in the numbers of both unipotential granulocytic and erythrocytic stem cells is compatible with these observations and implies that hematopoietic stem cells may have a basic rate of division that is in part independent of stimuli to differentiate. The implications of these findings for the possible modification of antineoplastic drug-induced bone marrow toxicity are discussed.     

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.     

Role of the MRP1/ABCC1 multidrug transporter protein in cancer

Multidrug resistance is a major obstacle to cancer treatment and leads to poor prognosis for the patient. Multidrug resistance-associated protein 1 (MRP1) transports a wide range of therapeutic agents as well as diverse physiological substrates and may play a role in the development of drug resistance in several cancers including those of the lung, breast and prostate, as well as childhood neuroblastoma. The majority of patients with neuroblastoma present with widely disseminated disease at diagnosis and despite intensive treatment, the prognosis for such patients is dismal. There is increasing evidence that MRP1 is a MYCN target gene involved in the development of multidrug resistance in neuroblastoma. Given the importance of MRP1 overexpression in neuroblastoma, MRP1 inhibition may be a clinically relevant approach to improving patient outcome in this disease.     

Characterization of testudine melanomacrophage linear,membrane extension processes—Cablepodia—By phase and atomic force microscopy

Summary Melanomacrophages (MMs) are a component of an internal, pigmented cell system in liver and splenic tissues of some fishes, anurans, and reptiles. The cells have been found in centers or aggregates in sinusoids and are associated with cells capable of producing a peptide cytokine and immunoglobulins. A unique cell extension process has been observed in turtle MMs placed into cell culture, and this process has been studied by light and atomic force microscopy. These structures, referred to as cablepodia, are uniquely straight, narrow, and unbranching and appear to originate from growth cones opposite lamellipodia. Cablepodia were found to connect with other turtle MMs and fibroblasts forming cell networks. Dividing fibroblasts to which a cablepodium attached ceased cell division. The observations collectively suggest that a principal reason for aggregations of MMs in internal organs of lower vertebrates in their ability to form interconnected networks of cell processes for trapping and processing of particulate matter, cells and infectious organisms and, possibly, for the communication of cell signals and transfer of intracellular materials.     

Regulation of β-catenin trafficking to the membrane in living cells

β-catenin is a key mediator of the Wnt signaling process and accumulates in the nucleus and at the membrane in response to Wnt-mediated inhibition of GSK-3β. In this study we used live cell photobleaching experiments to determine the dynamics and rate of recruitment of β-catenin at membrane adherens junctions (cell adhesion) and membrane ruffles (cell migration). First, we confirmed the nuclear-cytoplasmic shuttling of GFP-tagged β-catenin, and found that a small mobile pool of β-catenin can move from the nucleus to membrane ruffles in NIH 3T3 fibroblasts with a t_0.5 of ~ 30 s. Thus, β-catenin can shuttle between the nucleus and plasma membrane. The localized recruitment of β-catenin-GFP to membrane ruffles was more rapid, and the strong recovery observed after bleaching (mobile fraction 53%, t_0.5 ~5 s) is indicative of high turnover and transient association. In contrast, β-catenin-GFP displayed poor recovery at adherens junctions in MDCK epithelial cells (mobile fraction 10%, t_0.5 ~8 s), indicating stable retention at these membrane structures. We previously identified IQGAP1 as an upstream regulator of β-catenin at the membrane, and this is supported by photobleaching assays which now reveal IQGAP1 to be more stably anchored at membrane ruffles than β-catenin. Further analysis showed that LiCl-mediated inactivation of the kinase GSK-3β increased β-catenin membrane ruffle staining; this correlated with a faster rate of recruitment and not increased membrane retention of β-catenin. In summary, β-catenin displays a high turnover rate at membrane ruffles consistent with its dynamic internalization and recycling at these sites by macropinocytosis.     

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.     

Contraceptive potential of antibodies to the zona pellucida

The notion of a contraceptive vaccine based on gamete-specific surface antigens was first proposed over a decade ago, as the result of in-vitro and in-vivo studies, and in recent years has been the subject of intensive research. In particular, the zona pellucida has attracted much attention as a potential target for immunological intervention in the fertilization process. Such is the rapidly-expanding nature of research into the biochemical and biological characterization of this structure, that a review of the implications for the development of a contraceptive vaccine seems timely.     

Purification, sequencing and functions of calreticulin from maize

The most abundant proteins in the lumen of the endoplasmic reticulum(ER) are thought to be molecular chaperones, some of which mightalso be involved in calcium storage and release. We have purifiedcalreticulin from maize by ion exchange and reverse-phase chromatography.Identity with plant and animal calreticulins was confirmed byN-terminal amino acid sequencing and it was shown to bind calciumwith a calcium overlay technique. An antiserum raised to thepurified protein was used to screen an expression library andthe full coding sequence for maize calreticulin was determinedfrom the clones selected. The sequence shows 96% identity tobarley calreticulin and 55% identity to animal calreticulins.The three major functional regions are conserved, as are targetingand retention features. When visualized by indirect immunofluorescencemicroscopy, calreticulin was found to be confined to the ERand nuclear envelope of maize root cells. It was distributedthroughout the ER compartment and we found no evidence of calreticulin-enriched areas of ER, such as might be associated with specializedcalcium storage domains. Increasing or decreasing extracellularcalcium did not induce measurable changes in calreticulin levels.In addition, maize calreticulin, as well as other recognizedchaperones, was shown to bind to denatured protein and couldbe eluted specifically by nucleoside trisphosphates. Key words: Endoplasmic reticulum, calcium-binding protein, immunofluorescence, targeting, Zea mays L