Oncogenic RAS induces accelerated transition through G2/M and promotes defects in the G2 DNA damage and mitotic spindle checkpoints

Activating mutations of RAS are prevalent in thyroid follicular neoplasms, which commonly have chromosomal losses and gains. In thyroid cells, acute expression of HRAS(V12) increases the frequency of chromosomal abnormalities within one or two cell cycles, suggesting that RAS oncoproteins may interfere with cell cycle checkpoints required for maintenance of a stable genome. To explore this, PCCL3 thyroid cells with conditional expression of HRAS(V12) or HRAS(V12) effector mutants were presynchronized at the G(1)/S boundary, followed by activation of expression of RAS mutants and release from the cell cycle block. Expression of HRAS(V12) accelerated the G(2)/M phase by approximately 4 h and promoted bypass of the G(2) DNA damage and mitotic spindle checkpoints. Accelerated passage through G(2)/M and bypass of the G(2) DNA damage checkpoint, but not bypass of the mitotic spindle checkpoint, required activation of mitogen-activated protein kinase (MAPK). However, selective activation of the MAPK pathway was not sufficient to disrupt the G(2) DNA damage checkpoint, because cells arrested appropriately in G(2) despite conditional expression of HRAS(V12,S35) or BRAF(V600E). By contrast to the MAPK requirement for radiation-induced G(2) arrest, RAS-induced bypass of the mitotic spindle checkpoint was not prevented by pretreatment with MEK inhibitors. These data support a direct role for the MAPK pathway in control of G(2) progression and regulation of the G(2) DNA damage checkpoint. We propose that oncogenic RAS activation may predispose cells to genomic instability through both MAPK-dependent and independent pathways that affect critical checkpoints in G(2)/M.     

In Vitro Priming Recapitulates In Vivo HIV-1 Specific T Cell Responses,Revealing Rapid Loss of Virus Reactive CD4+ T Cells in Acute HIV-1 Infection

Background

The requirements for priming of HIV-specific T cell responses initially seen in infected individuals remain to be defined. Activation of T cell responses in lymph nodes requires cell-cell contact between T cells and DCs, which can give concurrent activation of T cells and HIV transmission.

Methodology

The study aim was to establish whether DCs pulsed with HIV-1 could prime HIV-specific T cell responses and to characterize these responses. Both infectious and aldrithiol-2 inactivated noninfectious HIV-1 were compared to establish efficiencies in priming and the type of responses elicited.

Findings

Our findings show that both infectious and inactivated HIV-1 pulsed DCs can prime HIV-specific responses from naïve T cells. Responses included several CD4⁺ and CD8⁺ T cell epitopes shown to be recognized in vivo by acutely and chronically infected individuals and some CD4⁺ T cell epitopes not identified previously. Follow up studies of acute and recent HIV infected samples revealed that these latter epitopes are among the earliest recognized in vivo, but the responses are lost rapidly, presumably through activation-induced general CD4⁺ T cell depletion which renders the newly activated HIV-specific CD4⁺ T cells prime targets for elimination.

Conclusion

Our studies highlight the ability of DCs to efficiently prime naïve T cells and induce a broad repertoire of HIV-specific responses and also provide valuable insights to the pathogenesis of HIV-1 infection in vivo.     

Breaking the diffraction barrier: super-resolution imaging of cells

Anyone who has used a light microscope has wished that its resolution could be a little better. Now,?after centuries of gradual improvements, fluorescence microscopy has made a quantum leap in its resolving power due, in large part, to advancements over the past several years in a new area of research called super-resolution fluorescence microscopy. In this Primer, we explain the principles of various super-resolution approaches, such as STED, (S)SIM, and STORM/(F)PALM. Then, we describe recent applications of super-resolution microscopy in cells, which demonstrate how these approaches are beginning to provide new insights into cell biology, microbiology, and neurobiology.     

An overview of the serpin superfamily

Serpins are a broadly distributed family of protease inhibitors that use a conformational change to inhibit target enzymes. They are central in controlling many important proteolytic cascades, including the mammalian coagulation pathways. Serpins are conformationally labile and many of the disease-linked mutations of serpins result in misfolding or in pathogenic, inactive polymers.     

Discrete regional distributions suggest diverse functional roles of calcium channel alpha1 subunits in sperm

The Ca channels of male germ-line cells are partially characterized, but the molecular properties and subcellular localization of the Ca channels of mature sperm are unknown. Here, we probe rodent sperm with anti-peptide antibodies directed to cytosolic domains of cloned rat brain alpha1A, alpha1C, and alpha1E Ca channel subunits. Each recognizes a 200- to 245-kDa band on immunoblots of whole rat sperm extracts. A smaller ( approximately 110-kDa) alpha1C band also is detected. Confocal fluorescence images of mouse sperm show characteristic patterns of punctate alpha1A-, alpha1C-, and alpha1E-immunoreactivity. For alpha1A, the puncta are larger, less numerous, and more variable in distribution than for alpha1C and alpha1E. They are absent from the acrosomal crescent, but are present elsewhere over the sperm head, often at the apical tip and equatorial segment. They also are found at irregular intervals along both the midpiece and the principal piece of the flagellum. For alpha1C and alpha1E, puncta are dense along dorsal and ventral aspects of the acrosomal cap. For alpha1E but not alpha1C, the remainder of the acrosomal region also is labeled. Neither is found in the postacrosomal region or on the midpiece. Puncta of alpha1C and alpha1E occur at regular intervals each in two parallel rows, at the dorsal and ventral aspects of the proximal segment of the flagellar principal piece. The puncta in these arrays become less abundant and intense in the distal flagellum. These results demonstrate that multiple Ca channel proteins are present in mature sperm and are regionally localized in ways that may give them different regulatory roles.     

A genetic screen for synaptic transmission mutants mapping to the right arm of chromosome 3 in Drosophila

Neuronal function depends upon the proper formation of synaptic connections and rapid communication at these sites, primarily through the regulated exocytosis of chemical neurotransmitters. Recent biochemical and genomic studies have identified a large number of candidate molecules that may function in these processes. To complement these studies, we are pursuing a genetic approach to identify genes affecting synaptic transmission in the Drosophila visual system. Our screening approach involves a recently described genetic method allowing efficient production of mosaic flies whose eyes are entirely homozygous for a mutagenized chromosome arm. From a screen of 42,500 mutagenized flies, 32 mutations on chromosome 3R that confer synaptic transmission defects in the visual system were recovered. These mutations represent 14 complementation groups, of which at least 9 also appear to perform functional roles outside of the eye. Three of these complementation groups disrupt photoreceptor axonal projection, whereas the remaining complementation groups confer presynaptic defects in synaptic transmission without detectably altering photoreceptor structure. Mapping and complementation testing with candidate mutations revealed new alleles of the neuronal fate determinant svp and the synaptic vesicle trafficking component lap among the collection of mutants recovered in this screen. Given the tools available for investigation of synaptic function in Drosophila, these mutants represent a valuable resource for future analysis of synapse development and function.     

Analytical procedures for the quantification of isotopic amino acid incorporation into photosynthetic proteins of Synechocystis PCC 6803

The mechanism of oxygen evolution has been an enigma for nearly two centuries. Pioneering work by Bessel Kok, Pierre Joliot, and many others during the last quarter century has provided valuable insight into this most unique and important chemical reaction. The late 1970s and early 1980s saw the introduction of biochemical techniques for the purification of photosynthetic complexes that have, in turn, stimulated the biophysical chemists and spectroscopists to apply high resolution techniques in order to resolve the structure/function relationships in these protein complexes. Valuable information about events at the atomic level can be gained through isotopic substitution of particular amino acids thought to be important in the catalytic process. The ability to generate functional auxotrophs in the photosynthetic cyanobacterium Synechocystis 6803 has been used successfully to identify the redox active components Z and D as tyrosine residues in the reaction center of Photosystem II. In this report, we present results of the application of specific isotopic labeling for high resolution spectroscopy of purified PS II particles. We have developed analytical procedures for monitoring the incorporation of both ²H and ¹⁷O labeled amino acids by gas chromatography-mass spectroscopic analysis. We also show that the growth curve of cells subjected to obligate auxotrophy displays two distinct stationary phases; one that corresponds to depletion of exogenous amino acids, and a second that corresponds to the normal cell density at stationary phase. Cells harvested at the second stationary phase show little or no retention of the labeled amino acid.Abbreviations D1 D2 reaction center core proteins of Photosystem II encoded by the psbA and psbD genes, respectively - FTIR Fourier transform infrared - ENDOR electron-nuclear double resonance - ESEEM electron spin-echo envelope modulation - EXAFS extended X-ray absorption fine structure - iBuCF isobutylchloroformate - OD optical density - XANES X-ray absorption near-edge structure - Y_D Y_Z redox active tyrosines of PS II     

Enrichment and kinetics of biodegradation of 1-naphthylamine in activated sludge

Sequencing-batch reactors were used to develop an activated sludge enrichment culture capable of degrading 1-naphthylamine (1NA). Approximately 5 months acclimation with salicylic acid (1600 mg l^–1) as the primary source of carbon were required to obtain an enrichment culture able to degrade even small quantities of 1NA. After an additional 4 months acclimation, during which the concentration of salicyclic acid was decreased to 50 mg l^–1, a culture developed that degraded 1NA concentrations as high as 300 mg l^–1. Kinetic determinations showed that 1NA degradation (in the presence of salicylate) followed Michaelis-Menten kinetics with K _m and V _m values of 32.5±2.2 mg l^–1 and 375±18 ng 1NA mg^–1 cells h^–1, respectively. The same enrichement was able to degrade 1NA when present as the sole source of carbon and energy and to convert approximately 87% to CO₂.     

Antigen-induced murine B cell lymphomas. II. Exploitation of the surface idiotype as tumor specific antigen.

In the accompanying report, we have described the characterization of two unusual murine B cell lymphomas, CH1 and CH2. A heterologous antiserum, which we refer to as "anti-idiotype" serum, has been raised to the detergent-solubilized surface immunoglobulin of CH1. The following criteria have established that this antiserum is specific for the CH1 tumor and that it reacts with V region determinants of the tumor surface IgM: 1) the antiserum reacts with CH1 tumor cells, but not normal mouse lymphoid cells or CH2 tumor cells, in indirect immunofluorescence and C-dependent cytotoxicity testing, 2) capping with the anti-idiotype serum removes all or most of the tumor surface Ig, 3) the antiserum forms a single band of precipitation against serum from CH1 tumor-bearing mice, when tested by double diffusion precipitin analysis, and 4) a single band of precipitation is formed in the electrophoretic migration position of IgM when the anti-idiotype antiserum is tested against serum from CH1 tumor-bearing mice in immunoelectrophoresis. Furthermore, we have demonstrated that this antiserum is useful in monitoring tumor growth and is a potent immunotherapeutic agent. Specifically, 50% of mice injected with a lethal tumor inoculum and given a small dose of anti-idiotype serum 2 days later remain tumor free, whereas all tumor-challenged control mice died within 30 days.