Localization of estrogen receptors in uterine cells. An appraisal of translocation.

The nuclear localization of estrogen receptors has been examined under conditions which minimize redistribution and localization artifacts. A procedure is presented which rapidly lyses suspensions of cells from immature rat uteri by using 0.04% Triton X-100 in isotonic buffer. The ‘nuclei’ which are obtained after lysis have a median diameter of 1μm and are devoid of nuclear membranes. There is close agreement between the number of cells before lysis and the number of nuclear particles after lysis. Triton X-100 gave no interference with quantitative binding of estradiol to receptor and no alteration in the sedimentation behavior of receptor on sucrose gradients containing high or low salt. Using this procedure to monitor the dynamics of estrogen receptor distribution within uterine cells after exposure to estradiol, translocation of estrogen receptor to the nucleus was observed to occur at a rate slightly slower than the rate at which estradiol was specifically bound to free cells or receptors. The difference in these rates is compatible with a model in which estradiol must first bind to the receptor before the binding complex moves to the nucleus. The rate of nuclear translocation was temperature-dependent and was observed to occur at 0 °C, provided that enough time was allowed for steroid entry, receptor charging and transit to the nucleus. Two distinct phases were observed to characterize nuclear receptor localization. In the first phase after hormone exposure, estrogen receptor progressively accumulated in the nucleus; afterwards, estrogen receptor was progressively lost from the nucleus but could not be detected in other subcellular compartments in a form still binding hormone. Since high cell viability was maintained during these manipulations, loss of nuclear receptor was not due to cell damage during in vitro incubation. These studies suggest that this decline in nuclear receptor level after hormone interaction, which is known to occur in vivo, may be a normal event during estrogen interaction with target cells.     

Polyanion induced physical changes in isolated nuclei

Small angle light scattering techniques were used to determine the effects of selected polyanions on isolated mouse spleen nuclei. The light scattering measurements made it possible to distinguish nuclear swelling from lysis and/or aggregation. Heparin and dextran sulfate were shown to induce nuclear swelling. Chondroitin sulfate, although structurally similar to heparin, had no observable effect on the nuclei. Polystyrene sulfonate and pyran copolymer caused nuclear aggregation and lysis, respectively.     

In-cell NMR spectroscopy

Structural studies by in-cell nuclear magnetic resonance are a developing new field of research, and their objective is to obtain structural information of proteins and other biological macromolecules in the cytoplasm of Escherichia coli cells. The major limitation of in-cell experiments is cell lysis that occurs during the experiments. In this article, we describe how inhibition of autologous expression by rifampicin at a high concentration decreases cell lysis in E. coli. We suggest that rifampicin is acting in the programmed cell death gene system MazEF, which is triggered by stress conditions and ultimately leads to cell lysis.     

Resource dynamics during infection of Micromonas pusilla by virus MpV-Sp1

Viruses infecting marine phytoplankton drive phytoplankton diversity, terminate blooms and shuttle genetic material. Assessments of the scale of viral impacts on trophic networks are, however, speculative. We investigated fluxes of DNA between host and virus during infection of the prasinophyte alga Micromonas pusilla by phycodnavirus MpV SP1. Under a light-dark regimen, viral genomes accumulated to a transient peak within 24 h, at the expense of both host DNA synthesis and nuclear DNA. Viral genome abundance then declined soon after host lysis. This release of a phosphate-rich nucleotide pool during viral infection of phytoplankton should be considered in trophic models. Lysis required light and was stalled in darkness, meanwhile viral genome replication proceeded slowly in the dark. Viral exploitation of this host is therefore only partially light-dependent and infected phytoplankton are poised to lyse at dawn or if mixed to the photic zone. The chloroplast genome remained intact until lysis, indicating that either this DNA pool is inaccessible or the virus spares the chloroplast for its energy and reductant generation. The photochemical turnover of residual Photosystem II complexes accelerated during lysis, indicating that events in late infection heighten demands on the remaining host photosynthetic systems, consistent with the light dependency of lysis.     

Affinity purification of influenza virus ribonucleoprotein complexes from the chromatin of infected cells

Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the single-stranded genome is encapsidated by the nucleoprotein (NP), and associated with the trimeric polymerase complex consisting of the PA, PB1, and PB2 subunits. However, in contrast to most RNA viruses, influenza viruses perform viral RNA synthesis in the nuclei of infected cells. Interestingly, viral mRNA synthesis uses cellular pre-mRNAs as primers, and it has been proposed that this process takes place on chromatin. Interactions between the viral polymerase and the host RNA polymerase II, as well as between NP and host nucleosomes have also been characterized. Recently, the generation of recombinant influenza viruses encoding a One-Strep-Tag genetically fused to the C-terminus of the PB2 subunit of the viral polymerase (rWSN-PB2-Strep) has been described. These recombinant viruses allow the purification of PB2-containing complexes, including vRNPs, from infected cells. To obtain purified vRNPs, cell cultures are infected, and vRNPs are affinity purified from lysates derived from these cells. However, the lysis procedures used to date have been based on one-step detergent lysis, which, despite the presence of a general nuclease, often extract chromatin-bound material only inefficiently. Our preliminary work suggested that a large portion of nuclear vRNPs were not extracted during traditional cell lysis, and therefore could not be affinity purified. To increase this extraction efficiency, and to separate chromatin-bound from non-chromatin-bound nuclear vRNPs, we adapted a step-wise subcellular extraction protocol to influenza virus-infected cells. Briefly, this procedure first separates the nuclei from the cell and then extracts soluble nuclear proteins (here termed the "nucleoplasmic" fraction). The remaining insoluble nuclear material is then digested with Benzonase, an unspecific DNA/RNA nuclease, followed by two salt extraction steps: first using 150 mM NaCl (termed "ch150"), then 500 mM NaCl ("ch500") (Fig. 1). These salt extraction steps were chosen based on our observation that 500 mM NaCl was sufficient to solubilize over 85% of nuclear vRNPs yet still allow binding of tagged vRNPs to the affinity matrix. After subcellular fractionation of infected cells, it is possible to affinity purify PB2-tagged vRNPs from each individual fraction and analyze their protein and RNA components using Western Blot and primer extension, respectively. Recently, we utilized this method to discover that vRNP export complexes form during late points after infection on the chromatin fraction extracted with 500 mM NaCl (ch500).     

小鼠体细胞核移植程序的研究

采用直接去核法、透明带切割法、PMM法3种核移植方法进行小鼠卵母细胞去核的研究。3种方法都未对卵母细胞核进行示踪,仅以第一极体作为参照进行去核,都属于盲吸法范畴,在去核率上没有显著差异。直接去核法对卵母细胞造成较大的伤害,去核操作过程中极易使卵母细胞膜破裂,发生崩解;透明带切割法分步操作使操作变得柔和,对卵母细胞的刺激减小,去核卵母细胞的存活率较高;PMM法靠脉冲电压在透明带上打孔进行辅助去核,脉冲参数稍大或压透明带太紧都极易在击破透明带的同时击破卵膜,使卵母细胞发生崩解。在构建重构胚的过程中,胞质内注射法较电融合法而言,程序简单,带入的供体胞质较少,构建重构胚的效率更高。     

The Simian virus 40 late viral protein VP4 disrupts the nuclear envelope for viral release

Simian virus 40 (SV40) appears to initiate cell lysis by expressing the late viral protein VP4 at the end of infection to aid in virus dissemination. To investigate the contribution of VP4 to cell lysis, VP4 was expressed in mammalian cells where it was predominantly observed along the nuclear periphery. The integrity of the nuclear envelope was compromised in these cells, resulting in the mislocalization of a soluble nuclear marker. Using assays that involved the cellular expression of VP4 or the treatment of cells with purified VP4, we found that the central hydrophobic domain and a proximal C-terminal nuclear localization signal of VP4 were required for (i) cytolysis associated with prolonged expression; (ii) nuclear envelope accumulation; and (iii) disruption of the nuclear, red blood cell, or host cell membranes. Furthermore, a conserved proline within the hydrophobic domain was required for membrane perforation, suggesting that this residue was crucial for VP4 cytolytic activity. These results indicate that VP4 forms pores in the nuclear membrane leading to lysis and virus release.     

A rapid method for preparation of nuclear membranes from mammalian cells.

A simple, rapid procedure for disrupting nuclei of mammalian cells has been characterized. This procedure involves treating isolated nuclear suspensions with the natural polyanion heparin in 0.125 m sucrose buffered between pH 7.0 and 8.0. The rate and extent of nuclear lysis are dependent upon the ratio of nuclei concentration to heparin concentration. This procedure avoids use of intense mechanical disruption, enzymatic digestion, and high salt concentrations for achieving optimum lysis of the nuclei. This method can also be used for large-scale nuclear membrane preparations.     

Cross Talk between NDR Kinase Pathways Coordinates Cytokinesis with Cell Separation in Schizosaccharomyces pombe

NDR (nuclear Dbf-2-related) kinases constitute key regulatory nodes in signaling networks that control multiple biological processes such as growth, proliferation, mitotic exit, morphogenesis, and apoptosis. Two NDR pathways called the septation initiation network (SIN) and the morphogenesis Orb6 network (MOR) exist in the fission yeast Schizosaccharomyces pombe. The SIN promotes cytokinesis, and the MOR drives cell separation at the end of cytokinesis and polarized growth during interphase. We showed previously that cross talk exists between these two pathways, with the SIN inhibiting the MOR during cytokinesis through phosphorylation of the MOR component Nak1 by the SIN Sid2 kinase. The reason for this inhibition remained uncertain. We show here that failure to inhibit MOR signaling during cytokinesis results in cell lysis at the site of septum formation. Time-lapse analysis revealed that MOR signaling during cytokinesis causes cells to prematurely initiate septum degradation/cell separation. The cell lysis phenotype is due to premature initiation of cell separation because it can be rescued by mutations in genes required for cell separation/septum degradation. We also shed further light on how the SIN inhibits the MOR. Sid2 phosphorylation of the MOR proteins Sog2 and Nak1 is required to prevent cell lysis during cytokinesis. Together, these results show that SIN inhibition of the MOR enforces proper temporal ordering of cytokinetic events.     

Investigating the two regimes of fibrin clot lysis: an experimental and computational approach

It has been observed in vitro that complete clot lysis is generally preceded by a slow phase of lysis during which the degradation seems to be inefficient. However, this slow regime was merely noticed, but not yet quantitatively discussed. In our experiments, we observed that the lysis ubiquitously occurred in two distinct regimes, a slow and a fast lysis regime. We quantified extensively the duration of these regimes for a wide spectrum of experimental conditions and found that on average, the slow regime lasts longer than the fast one, meaning that during most of the process, the lysis is ineffective. We proposed a computational model in which the properties of the binding of the proteins change during the lysis: first, the biochemical reactions take place at the surface of the fibrin fibers, then in the bulk, resulting in the observed fast lysis regime. This simple hypothesis appeared to be sufficient to reproduce with a great accuracy the lysis profiles obtained experimentally.     

Plasma membrane and nuclear envelope integrity during the blebbing stage of apoptosis: a time‐lapse study

Background information. The execution phase of apoptosis is characterized by extensive blebbing of the plasma membrane, which usually results in secondary lysis in vitro. To analyse the permeability of cellular membranes during this process, we induced apoptosis in human melanoma A375 cells that had been transfected with fluorescently tagged proteins which were targeted to different subcellular locations. Results. The dual treatment of resveratrol and butyrate produced a synergistic induction of apoptosis by blocking different phases of the cell cycle. Changes in the plasma membrane, nuclear envelope and nucleoli were monitored by time‐lapse confocal microscopy. Fluorescently labelled proteins were not mis‐localized from their original locations in any of the cells undergoing blebbing for several hours. Thus the maintenance of karyophilic and nucleolar proteins within the nucleus during the blebbing stage and the accessibility of vital selective chromatin dyes confirmed a functional preservation of the nuclear compartment until the final necrotic blister. The translocation of phosphatidylserine to the outer leaflet of the plasma membrane was not detected during the blebbing period. Conclusion. These results show that the functional integrity of the nuclear envelope and plasma membrane may be conserved until the end of the execution phase of apoptosis.     

The degenerating processes of germinal cells during oogenesis in Lithobius forticatus L. (Myriapoda chilopoda)]

The ovary of Lithobius forficatus always contains some degenerating oocytes. The cytologic evolution during the oocyte degenerescence is described. Cytoplasmic lysis takes place before nuclear lysis and the vitellinic reserves resist the longest. The degenerates coming from previtellogenetic or poorly vitellogenetic cells are essentially recovered by the follicular cells. If large vitellogenetic or mature oocytes are affected, their vitellinic reserves are released into the ovarian cavity and included in the cells of the ovarian epithelium. The number of degenerating oocytes is in relation with the external conditions; it reaches a maximum at the end of the spring. The temperature may act directly or indirectly through the endocrine centers.     

The antimicrobial effect of a structural variant of subtilin against outgrowing Bacillus cereus T spores and vegetative cells occurs by different mechanisms.

Subtilin is a ribosomally synthesized antimicrobial peptide that contains several unusual amino acids as a result of posttranslational modifications. Site-directed mutagenesis was employed to construct a structural variant of subtilin in which the unusual dehydroalanine (Dha) residue at position 5 was changed to alanine. Proton nuclear magnetic resonance spectroscopy, amino acid composition, and N-terminal sequence analysis established that the mutation did not disrupt posttranslational processing of the precursor peptide. This mutant subtilin was devoid of antimicrobial activity as assessed by its lack of inhibitory effects on outgrowth of Bacillus cereus T spores. However, this same mutant subtilin was fully active with respect to its ability to induce lysis of vegetative B. cereus T cells. Because an intact Dha-5 residue is required in the one instance but not in the other, it was concluded that the molecular mechanism by which subtilin inhibits (without lysis) spore outgrowth is not the same as the mechanism by which it inhibits (with lysis) vegetative cells.     

A NOVEL VIRUS (HaNIV) CAUSES LYSIS OF THE TOXIC BLOOM-FORMING ALGA HETEROSIGMA AKASHIWO (RAPHIDOPHYCEAE)

We describe a previously unknown virus that causes lysis of the toxic bloom-forming alga Heterosigma akashiwo (Hada) Hara et Chihara (Raphidophyceae). Heterosigma akashiwo nuclear inclusion virus (HaNIV) does not resemble other algal viruses described to date. HaNIV is small (ca. 30 nm diameter), is assembled in the nucleus, and forms crystalline arrays. We estimate that approximately 10⁵ HaNIV particles are released during lysis of a cell. During a time-course experiment, TEM revealed the first signs of HaNIV infection 24 h after viral addition, and by 74 h 98% of observed cells were visibly infected. The onset of cell lysis, as indicated by a decrease in the relative fluorescence of the cultures, was apparent by 42 h postinfection. The heterochromatin of infected cells is frequently found at the margin of the nucleoplasm, which is consistent with virus-mediated programmed cell death, or apoptosis. HaNIV is clearly different from other described viruses that infect algae, including other viral pathogens of H. akashiwo. These results indicate that viruses other than Phycodnaviridae are pathogens and cause mortality of microalgae in marine systems. It is likely that HaNIV plays an integral role in the population dynamics of H. akashiwo.     

Simian virus 40 late proteins possess lytic properties that render them capable of permeabilizing cellular membranes

Many nonenveloped viruses have evolved an infectious cycle that culminates in the lysis or permeabilization of the host to enable viral release. How these viruses initiate the lytic event is largely unknown. Here, we demonstrated that the simian virus 40 progeny accumulated at the nuclear envelope prior to the permeabilization of the nuclear, endoplasmic reticulum, and plasma membranes at a time which corresponded with the release of the progeny. The permeabilization of these cellular membranes temporally correlated with late protein expression and was not observed upon the inhibition of their synthesis. To address whether one or more of the late proteins possessed an inherent capacity to induce membrane permeabilization, we examined the permeability of Escherichia coli that separately expressed the late proteins. VP2 and VP3, but not VP1, caused the permeabilization of bacterial membranes. Additionally, VP3 expression resulted in bacterial cell lysis. These findings demonstrate that VP3 possesses an inherent lytic property that is independent of eukaryotic signaling or cell death pathways.     

Death and lysis ofEscherichia coli 15 TAU cells after pulse-interrupted thymine starvation

The loss of colony-forming ability and lysis of cells were studied after pulse-interrupted thymine starvation of a culture ofEscherichia coli 15 TAU which before starvation grew exponentially or was synchronized by starvation for arginine and uracil. Even a 5-minute pulse cancelled the effects of previous starvation leading to lysis, while loss of colony-forming ability after pulse was changed only slightly. The course of lysis and thymineless death after synchronization did not depend on the presence or absence of thymine during about a 40-min interval just after synchronization. With an appropriate time-schedule of pulses, it was possible to prolong the total time of interrupted starvation after which lysis did not appear.     

Kinetic and Morphological Observations on the Yeast Phase of Histoplasma capsulatum During Protoplast Formation

Protoplast formation by Histoplasma capsulatum yeasts using high concentrations of MgSO(4) occurs either by lysis of the bud or lysis of the entire cell wall. Both mechanisms may also occur simultaneously. Neither the protoplast emerging through a hole in the cell wall nor the freshly released protoplast has a recognizable cell wall or the remnant of such. The protoplast contains all the organelles of the normal cell except for mesosomes. During protoplast formation the nucleus increases in size and produces several nuclear masses by the invaginations of the internal layer of the nuclear membrane. All these nuclear masses are surrounded by the external layer of the nuclear membrane. Several nuclei with a normal nuclear membrane are formed later.     

Lysis of human keratinocytes by allogeneic HLA class I-specific cytotoxic T cells. Keratinocyte ICAM-1 (CD54) and T cell LFA-1 (CD11a/CD18) mediate enhanced lysis of IFN-gamma-treated keratinocytes.

Exposure of human KC to IFN-gamma increases their susceptibility to lysis by CTL. The mechanism of this enhanced lysis was investigated by analyzing interactions of IFN-gamma-treated and nontreated cultured KC with allogeneic class I-specific CTL clones. rIFN-gamma treatment augmented KC lysis in a time- and dose-dependent manner. Increased lysis of IFN-KC was detected after only 2 h of IFN-gamma treatment and was maximal by 12 h. Enhanced lysis of IFN-KC was Ag-specific, inasmuch as nonantigenic IFN-KC were not lysed either directly or as bystanders during the lysis of antigenic KC. Parallel immunofluorescence and cytotoxicity assays of KC treated with IFN-gamma for various intervals revealed a direct correlation between the degree of increased KC lysis and levels of cell surface ICAM-1 (CD54), but not of specific alloantigen or beta 2-microglobulin. Lysis of nontreated KC was blocked by mAb against class I or CD3, but not by mAb against ICAM-1 or LFA-1. In contrast, lysis of IFN-KC was partially inhibited by anti-ICAM-1 or anti-LFA-1 mAb, but resisted inhibition by anti-class I mAb except in the presence of anti-ICAM-1. These results indicate that both ICAM-1/LFA-1 and Ag/CD3-TcR interactions are important for Ag-specific lysis of IFN-KC, whereas lysis of nontreated KC depends on Ag/CD3-TcR but not ICAM-1/LFA-1 interactions. Equivalent inhibition of IFN-KC lysis by mAb against ICAM-1 or LFA-1 suggests that ICAM-1 is the only LFA-1 ligand involved in enhanced IFN-KC lysis. Furthermore, enhanced CTL lysis of KC after short-term IFN-gamma treatment can be explained solely on the basis of ICAM-1 induction, because all of the increase in specific lysis associated with IFN-gamma treatment could be blocked by mAb that block ICAM-1/LFA-1 interactions.     

In vivo crosslinking of nuclear proteins to DNA by cis-diamminedichloroplatinum (II) in differentiating rat myoblasts

When cells are briefly exposed to cis-diamminedichloroplatinum (II) before lysis in high sodium dodecyl sulfate-urea solutions, the high molecular-weight nucleic acids pelleted by ultracentrifugation contain an increased level of bound proteins when compared to a similar fraction from untreated cells. Subsequent shearing of the pelleted DNA followed by treatment with DNase permits electrophoretic and immunoblot analysis of the crosslinked proteins. In the present study such experiments were carried out with reference to nuclear envelope pore complex proteins in the differentiating L8 rat skeletal muscle cells. The results show that (i) whereas the major lamin proteins crosslinked to DNA in both myoblast and myotubes, lamin B is crosslinked to a greater extent to DNA in myotubes; (ii) a 62-kDa lectin-binding glycoprotein is apparently situated differently with respect to DNA in myotube nuclei; and (iii) the crosslinking pattern of the nuclear matrix proteins to DNA is qualitatively similar in myoblast and myotubes. In addition, lamin C', a modified form of lamin C, not observed in intact nonmuscle cells previously [Glass et al. (1985) J. Biol. Chem. 260, 1895-1900], exists as a native component of the nuclear lamina in rat skeletal myotubes but not in myoblasts. These results point to significant structural alterations in the proteins of the nuclear lamina-pore complex during myogenesis.     

Developmentally induced autolysis during fruiting body formation by Myxococcus xanthus.

The developmental events during fruiting body construction by the myxobacterium M. xanthus is an orderly process characterized by several sequential stages: growth leads to aggregation leads to formation of raised, darkened mounds of cells leads to autolysis leads to myxospore induction. The temporal sequence of autolysis followed by myxospore induction is consistent with the interpretation that developmental autolysis provides essential requirements for the surviving cells to induce to myxospores. At intermediate developmental times on agar plates a fraction of the cell population is irreversibly committed to lyse; i.e., lysis continues in liquid growth medium or in magnesium-phosphate buffer. Lysis is cell concentration independent and is therefore likely to be by an autolytic mechanism. The lysis sequence can be preliminarily characterized as having an early stage during which deoxyribonucleic acid synthesis continues and a later irreversible stage during which deoxyribonucleic acid synthesis does not occur. Irreversible lysis in liquid growth medium or in magnesium-phosphate buffer is initiated on agar plates during nutrient deprivation and such lysis results in the induction of a fraction of the population to myxospores. This induction is dependent upon the concentration of lysis products, thus providing evidence that developmentally induced autolysis is required for myxospore induction.