Acquisition of embryogenic competency does not require cell division in carrot somatic cell

Totipotency is the ability of a cell to regenerate the entire organism, even after previous differentiation as a specific cell. When totipotency is coupled with active cell division, it was presumed that cell division is essential for this expression. Here, using the stress-induction system of somatic embryos in carrots, we show that cell division is not essential for the expression of totipotency in somatic/embryonic conversion. Morphological and histochemical analyses showed that the cell did not divide during embryo induction. Inhibitors of cell division did not affect the rate of somatic embryo formation. Our results indicate that the newly acquired trait of differentiation appears without cell division, but does not arise with cell division as a newborn cell.     

Evidence that membrane transduction of oligoarginine does not require vesicle formation

The involvement of vesicular formation processes in the membrane transduction and nuclear transport of oligoarginine is currently a subject of controversy. In this report, a novel quantitative method which allows for the selective measurement of membrane transduction excluding concurrent endocytosis was used to determine the effects of temperature, endosomal acidification, endosomolysis, and several known inhibitors of endocytic pathways on the internalization of oligoarginine. The results show that, unlike endocytosis, transduction of oligoarginine was not affected by incubation at 16 degrees C as compared to the 37 degrees C control, and was only partially inhibited at 4 degrees C incubation. Additionally, membrane transduction was not inhibited to the same extent as endocytosis following treatment with ammonium chloride, hypertonic medium, amiloride, or filipin. The endosomolytic activity of oligoarginine was investigated by examining the leakage of FITC-dextran into the cytosolic compartment, which was not higher in the presence of oligoarginine. Furthermore, ammonium chloride showed no effect on the nuclear transport of oligoarginine. The data presented in this report indicate that membrane transduction is likely to occur at the plasma membrane without the formation of membrane vesicles, and the nuclear localization involves membrane transduction, rather than endocytosis of oligoarginine.     

Stimulation of pituitary gonadotropin release does not require internalization of gonadotropin-releasing hormone

Binding of gonadotropin-releasing hormone (GnRH, pyro-Glu1-His2-Trp3-Ser4-Tyr5-Gly6-Leu7-Arg8-Pro9-Gly-NH210) to its plasma membrane receptor is the first step leading to the release of pituitary luteinizing hormone. As in the case of other plasma membrane receptors, patching, capping, and internalization of this hormone-receptor complex occurs rapidly following exposure of cultured pituitary cells to physiological levels of releasing hormone. In the present study we sought to determine whether gonadotropin release could occur under conditions which rigorously excluded internalization. A GnRH analog, D-Lys6-GnRH (to which a small quantity of [125I]iodoTyr5-D-Lys6-GnRH was added), was coupled by its epsilon-amino group with an N-hydroxysuccinimide ester then, through a 10-A spacer arm, to a cross-linked agarose matrix. Exposure of the product to proteases, soaps, detergents, solvents, chaotropic agents, or cell cultures resulted in dissociation of < 0.28% of biologically active releasing hormone. The apparent potency of the immobilized analog was one-fourth that of the free form and it was still capable of evoking a full luteinizing hormone secretory response. It can, therefore, be concluded that internalization of GnRH is not required for gonadotropin release.     

Induction of caspase-dependent apoptosis in cultured rat oligodendrocytes by murine coronavirus is mediated during cell entry and does not require virus replication

Murine coronavirus mouse hepatitis virus (MHV) causes demyelination of the central nervous system (CNS) in rats and mice. Apoptotic oligodendrocytes have been detected in the vicinity of the CNS demyelinating lesions in these animals. However, whether MHV can directly induce oligodendrocyte apoptosis has not been documented. Here, we established a rat oligodendrocyte culture that is morphologically and phenotypically indistinguishable from the primary rat oligodendrocytes. Using this culture, we showed that mature rat oligodendrocytes were permissive to MHV infection but did not support productive virus replication. Significantly, oligodendrocytes infected with both live and ultraviolet light-inactivated viruses underwent apoptosis to a similar extent, which was readily detectable at 24 h postinfection as revealed by apoptotic bodies and DNA fragmentation, indicating that MHV-induced apoptosis is mediated during the early stages of the virus life cycle and does not require virus replication. Prior treatment of cells with the lysosomotropic agents NH(4)Cl and chloroquine as well as the vacuolar proton pump-ATPase inhibitor bafilomycin A1, all of which block the acidification of the endosome, prevented oligodendrocytes from succumbing to apoptosis induced by MHV mutant OBLV60, which enters cells via endocytosis, indicating that fusion between the viral envelope and cell membranes triggers the apoptotic cascade. Treatment with the pan-caspase inhibitor Z-VAD-fmk blocked MHV-induced apoptosis, suggesting an involvement of the caspase-dependent pathway. Our results, thus, for the first time provide unequivocal evidence that infection of oligodendrocytes with MHV directly results in apoptosis. This finding provides an explanation for the destruction of oligodendrocytes and the damage of myelin sheath in MHV-infected CNS and suggests that oligodendrocyte apoptosis may be one of the underlying mechanisms for the pathogenesis of MHV-induced demyelinating diseases in animals.     

The release of C5a in complement-activated serum does not require C6

The influence of terminal complement components on the generation and release of the complement C5a fragment was investigated by comparing the levels of C5a in complement-activated serum with the levels of C5a produced in serum depleted of complement C6. In order to investigate the release of C5a, a modified C5a assay was developed that utilizes an anti-C5b monoclonal antibody to remove C5, C5b, and C5b-C5a complexes from samples prior to C5a assay. The modified assay was developed because the standard methodology, which includes an acid-precipitation step designed to dissociate C5a and C5b, cannot distinguish free C5a from the C5a that is bound to C5b. Therefore, the standard methodology is not capable of monitoring the influence of terminal components on C5a/C5b dissociation. Levels of C5a were measured in complement-activated whole human serum, in serum depleted of C6, and in serum containing inhibitory levels of anti-C6 Fab using both the modified C5a assay and the standard methodology. Sera were complement-activated with either zymosan to activate the alternative complement pathway or with antibody-coated sheep erythrocytes to activate the classical pathway. The levels of free C5a in C6-depleted sera after activation were equivalent to the C5a levels in activated whole serum, indicating that C6 is not required for the release of C5a from C5b. In addition, the quantity of C5a detected in zymosan-activated sera using the standard acid-precipitation methodology was greater than C5a levels when assayed using the modified immunoadsorption technique, confirming that acid-treatment enhances the C5a dissociation and promotes C5a recovery. Since the other terminal components, C7, C8, and C9, bind to C5b only after C5b only after C6 is bound, these results indicate that none of the terminal components are required for the release of C5a. Although the terminal components could influence the rate of C5a release, the quantity of C5a released in serum was entirely independent of terminal components.     

Some remarks on the movement of chromosomes during cell division

The possibility of drawing conclusions about the nature of the forces acting upon the chromosomes during division from observation of the rates of their movement in anaphase is pointed out. Some available data are discussed, and shown to agree quantitatively with the assumption that during anaphase the chromosomes are pulled apart by contracting elastic fibers.     

GFP tagging of budding yeast chromosomes reveals that protein–protein interactions can mediate sister chromatid cohesion

Background Precise control of sister chromatid separation is essential for the accurate transmission of genetic information. Sister chromatids must remain linked to each other from the time of DNA replication until the onset of chromosome segregation, when the linkage must be promptly dissolved. Recent studies suggest that the machinery that is responsible for the destruction of mitotic cyclins also degrades proteins that play a role in maintaining sister chromatid linkage, and that this machinery is regulated by the spindle-assembly checkpoint. Studies on these problems in budding yeast are hampered by the inability to resolve its chromosomes by light or electron microscopy.Results We have developed a novel method for visualizing specific DNA sequences in fixed and living budding yeast cells. A tandem array of 256 copies of the Lac operator is integrated at the desired site in the genome and detected by the binding of a green fluorescent protein (GFP)–Lac repressor fusion expressed from the HIS3 promoter. Using this method, we show that sister chromatid segregation precedes the destruction of cyclin B. In mad or bub cells, which lack the spindle-assembly checkpoint, sister chromatid separation can occur in the absence of microtubules. The expression of a tetramerizing form of the GFP–Lac repressor, which can bind Lac operators on two different DNA molecules, can hold sister chromatids together under conditions in which they would normally separate.Conclusions We conclude that sister chromatid separation in budding yeast can occur in the absence of microtubule-dependent forces, and that protein complexes that can bind two different DNA molecules are capable of holding sister chromatids together.     

Induction of secondary cytotoxic T-lymphocytes in vitro does not require cell proliferation.

Using a mouse in vitro allograft model, evidence has been obtained that, in contrast to the accepted view, the generation of cytotoxic effector function in T-lymphocytes does not necessarily require cell division.     

RpoS proteolysis is regulated by a mechanism that does not require the SprE (RssB) response regulator phosphorylation site

In Escherichia coli the response regulator SprE (RssB) facilitates degradation of the sigma factor RpoS by delivering it to the ClpXP protease. This process is regulated: RpoS is degraded in logarithmic phase but becomes stable upon carbon starvation, resulting in its accumulation. Because SprE contains a CheY domain with a conserved phosphorylation site (D58), the prevailing model posits that this control is mediated by phosphorylation. To test this model, we mutated the conserved response regulator phosphorylation site (D58A) of the chromosomal allele of sprE and monitored RpoS levels in response to carbon starvation. Though phosphorylation contributed to the SprE basal activity, we found that RpoS proteolysis was still regulated upon carbon starvation. Furthermore, our results indicate that phosphorylation of wild-type SprE occurs by a mechanism that is independent of acetyl phosphate.     

Externalization of phosphatidylserine during apoptosis does not specifically require either isoform of phosphatidylserine synthase

Phosphatidylserine (PtdSer) is made in mammalian cells by two PtdSer synthases, PSS1 and PSS2. In the plasma membrane PtdSer is normally localized on the inner leaflet but undergoes transbilayer movement during apoptosis and becomes exposed on the cell surface. We induced apoptosis with staurosporine in four Chinese hamster ovary (CHO) cell lines that are deficient in PSS1 and/or PSS2 to determine if PtdSer generated by either of these enzymes is required for externalization on the cell surface during apoptosis. The onset of apoptosis was confirmed by the appearance of morphological changes and DNA fragmentation while the plasma membrane remained largely intact. In all cell lines, regardless of their content of PSS1 and/or PSS2, apoptosis occurred to approximately the same extent, and within approximately the same time frame, as in parental CHO-K1 cells. The exposure of PtdSer on the cell surface was assessed by annexin V labeling and flow cytometry. Cells that were deficient in either PSS1 or PSS2, as well as cells that were deficient in both PSS1 and PSS2, externalized normal amounts of PtdSer. Our study demonstrates, that reduction of in vitro serine-exchange activity, even by 97%, does not restrict the externalization of PtdSer during apoptosis. Moreover, a normal level of expression of PSS1 and/or PSS2 is not required for generating the pool of PtdSer externalized during apoptosis.     

Glycoprotein 120 binding to CXCR4 causes p38-dependent primary T cell death that is facilitated by, but does not require cell-associated CD4

HIV-1 infection causes the depletion of host CD4 T cells through direct and indirect (bystander) mechanisms. Although HIV Env has been implicated in apoptosis of uninfected CD4 T cells via gp120 binding to either CD4 and/or the chemokine receptor 4 (CXCR4), conflicting data exist concerning the molecular mechanisms involved. Using primary human CD4 T cells, we demonstrate that gp120 binding to CD4 T cells activates proapoptotic p38, but does not activate antiapoptotic Akt. Because ligation of the CD4 receptor alone or the CXCR4 receptor alone causes p38 activation and apoptosis, we used the soluble inhibitors, soluble CD4 (sCD4) or AMD3100, to delineate the role of CD4 and CXCR4 receptors, respectively, in gp120-induced p38 activation and death. sCD4 alone augments gp120-induced death, suggesting that CXCR4 signaling is principally responsible. Supporting that model, AMD3100 reduces death caused by gp120 or by gp120/sCD4. Finally, prevention of gp120-CXCR4 interaction with 12G5 Abs blocks p38 activation and apoptosis, whereas inhibition of CD4-gp120 interaction with Leu-3a has no effect. Consequently, we conclude that gp120 interaction with CXCR4 is required for gp120 apoptotic effects in primary human T cells.     

Platelet-derived growth factor receptor inducibility is acquired immediately after translation and does not require glycosylation

Antibodies to phosphotyrosine were used in immunoprecipitation experiments to determine if post-translational modification of the platelet-derived growth factor (PDGF) receptor was required for the acquisition of ligand-induced tyrosine kinase activity. In intact Balb/c 3T3 fibroblasts, only the fully processed 180-kDa receptor was activated (tyrosine-phosphorylated) by PDGF. In a cell-free assay, however, the tyrosine-phosphorylated forms of the 160- and 145-kDa PDGF receptor precursors were also detected. These activated precursors were immunoprecipitated after brief (5-15 min) metabolic labeling periods. Thus the receptor could bind PDGF and induce tyrosine kinase activity shortly after translation. Unlike the mature form of the receptor, the 160-kDa receptor precursor was resistant to digestion with endo-alpha-N-acetylgalactosaminidase and thus did not contain O-linked oligosaccharides. Since this receptor precursor was activated by PDGF in the cell-free assay, the addition of O-linked sugars must not be necessary for ligand binding activity. Incubation of cells with tunicamycin completely inhibited N-linked glycosylation of the PDGF receptor. Nevertheless, PDGF still induced tyrosine phosphorylation of the 130-kDa aglycoreceptor in lysates of tunicamycin-treated cells. Thus, the addition of N-linked oligosaccharides was also not required for receptor activation. These findings show that the PDGF receptor acquired the ability to be activated by ligand cotranslationally or immediately after translation and that the addition of N- or O-linked oligosaccharides was not required for ligand binding and tyrosine kinase activities.     

FtsQ, FtsL and FtsI require FtsK, but not FtsN, for co-localization with FtsZ during Escherichia coli cell division

During cell division in Gram-negative bacteria, the cell envelope invaginates and constricts at the septum, eventually severing the cell into two compartments, and separating the replicated genetic materials. In Escherichia coli, at least nine essential gene products participate directly in septum formation: FtsA, FtsI, FtsL, FtsK, FtsN, FtsQ, FtsW, FtsZ and ZipA. All nine proteins have been localized to the septal ring, an equatorial ring structure at the division site. We used translational fusions to green fluorescent protein (GFP) to demonstrate that FtsQ, FtsL and FtsI localize to potential division sites in filamentous cells depleted of FtsN, but not in those depleted of FtsK. We also constructed translational fusions of FtsZ, FtsA, FtsQ, FtsL and FtsI to enhanced cyan or yellow fluorescent protein (ECFP or EYFP respectively), GFP variants with different fluorescence spectra. Examination of cells expressing different combinations of the fusions indicated that FtsA, FtsQ, FtsL and FtsI co-localize with FtsZ in filaments depleted of FtsN. These localization results support the model that E. coli cell division proteins assemble sequentially as a multimeric complex at the division site: first FtsZ, then FtsA and ZipA independently of each other, followed successively by FtsK, FtsQ, FtsL, FtsW, FtsI and FtsN.     

Induction of apoptosis by Sindbis virus occurs at cell entry and does not require virus replication

Sindbis virus (SV) is an alphavirus that causes encephalitis in mice and can lead to the apoptotic death of infected cells. To determine the step in virus replication during which apoptosis is triggered, we used UV-inactivated SV, chemicals that block virus fusion or protein synthesis, and cells that do and do not express heparan sulfate, the initial binding molecule for SV infection of many cells. In initial experiments, UV-inactivated neuroadapted SV (NSV) induced apoptosis in Chinese hamster ovary (CHO) cells lacking heparan sulfate in the presence of cycloheximide. When fusion of prebound UV-inactivated NSV was rapidly induced at the plasma membrane by exposure to acidic pH, apoptosis was induced in CHO cells with or without heparan sulfate in the presence or absence of cycloheximide in a virus dose-dependent manner. In N18 neuroblastoma cells, the relative virulence of the virus strain was an important determinant of apoptosis induced by UV-inactivated SV. Treatment of N18 cells with monensin to prevent endosomal acidification an hour before, but not 2 h after, exposure to live NSV blocked the induction of cell death, as did treatment with NH(4)Cl or bafilomycin A1. These studies indicate that SV can induce apoptosis at the time of fusion with the cell membrane and that virus replication is not required.     

Differentiation of Trypanosoma brucei may be stage non-specific and does not require progression of cell cycle

Ubiquitination and proteasomal degradation of cell cycle regulatory proteins are known to play a pivotal role in controlling the progression of the eukaryotic cell cycle. Using the technique of RNA interference (RNAi) on the bloodstream form of Trypanosoma brucei, we were able to knock down expression of each of the 11 non-ATPase regulatory subunit proteins (Rpns) in the 19S regulatory complex of the 26S proteasome. In each case, the knock-down led to arrest of cells within the G1 and G2 phases, suggesting blockage of cell cycle progression at both G1/S and G2/M boundaries. This finding differs from that observed previously in the procyclic form of T. brucei, in which loss of individual Rpns blocks only passage across the G2/M boundary. Thus, proteasomal degradation of additional regulatory protein(s) may be required for exiting from G1 phase in the bloodstream form. In vitro differentiation of each of the 11 Rpn-depleted bloodstream form cell lines into the procyclic form was monitored. Each cell line proceeded to completion of the differentiation process like the wild-type cells with the total percentage of differentiated cells about equivalent to the sum of G1 and G2 cells. Thus, cells trapped in either G1 or G2 phase can apparently still enter and complete the process of differentiation, which is probably neither stage specific nor dependent on the progression of the T. brucei cell cycle. The process is probably a simple pattern change of gene expression in the trypanosome induced by a temperature decrease from 37 degrees C to 26 degrees C in the presence of citrate and cis-aconitate.     

Hyperoxia-induced apoptosis does not require mitochondrial reactive oxygen species and is regulated by Bcl-2 proteins

Exposure of animals to hyperoxia results in lung injury that is characterized by apoptosis and necrosis of the alveolar epithelium and endothelium. The mechanism by which hyperoxia results in cell death, however, remains unclear. We sought to test the hypothesis that exposure to hyperoxia causes mitochondria-dependent apoptosis that requires the generation of reactive oxygen species from mitochondrial electron transport. Rat1a cells exposed to hyperoxia underwent apoptosis characterized by the release of cytochrome c, activation of caspase-9, and nuclear fragmentation that was prevented by the overexpression of Bcl-X(L.) Murine embryonic fibroblasts from bax(-/-) bak(-/-) mice were resistant to hyperoxia-induced cell death. The administration of the antioxidants manganese (III) tetrakis (4-benzoic acid) porphyrin, ebselen, and N-acetylcysteine failed to prevent cell death following exposure to hyperoxia. Human fibrosarcoma cells (HT1080) lacking mitochondrial DNA (rho(0) cells) that failed to generate reactive oxygen species during exposure to hyperoxia were not protected against cell death following exposure to hyperoxia. We conclude that exposure to hyperoxia results in apoptosis that requires Bax or Bak and can be prevented by the overexpression of Bcl-X(L). The mitochondrial generation of reactive oxygen species is not required for cell death following exposure to hyperoxia.     

Experimental evolution reveals that population density does not affect moth signalling behaviour and antennal morphology

Population density can play a vital role in determining investment in reproductive behaviours and morphologies of invertebrates. Males reared in high-density environments, where competition is high but difficulties in locating mates are low, may invest more in reproductive structures associated with sperm competition such as testes, at the expense of those traits associated with mate location, such as antennae. In species where females advertise for mates, such as most moths, a high-density environment may also lead to a reduction in pheromonal signalling (calling) length and frequency as a result of high mate abundance. While such responses have been shown at the phenotypically plastic level in moths, heritable evolutionary adaptations have seldom been tested, and studies of how population density influences pheromone signalling strategies are scarce. Here we use behavioural assays and scanning electron microscopic measurements to test whether larval population density influences, at the genetic level, the ability of males to locate females and male investment into antennal morphology, in addition to its effect on the frequency and duration of female calling. We used two replicated populations of the Indian meal moth Plodia interpunctella that had experimentally evolved under high or low population densities for 35 generations. We found no significant divergence in antennal morphology or mate acquisition behaviours between the two density populations. These findings suggest that although population density has the ability to create plastic changes in both morphological and behavioural traits, this factor alone is unlikely to be causing evolutionary change in male and female signalling in this species.     

L1-mediated colon cancer cell metastasis does not require changes in EMT and cancer stem cell markers

Aberrant activation of Wnt/β-catenin signaling is common in most sporadic and inherited colorectal cancer (CRC) cells leading to elevated β-catenin/TCF transactivation. We previously identified the neural cell adhesion molecule L1 as a target gene of β-catenin/TCF in CRC cells. Forced expression of L1 confers increased cell motility, invasion, and tumorigenesis, and the induction of human CRC cell metastasis to the liver. In human CRC tissue, L1 is exclusively localized at the invasive front of such tumors in a subpopulation of cells displaying nuclear β-catenin. We determined whether L1 expression confers metastatic capacities by inducing an epithelial to mesenchymal transition (EMT) and whether L1 cosegregates with cancer stem cell (CSC) markers. We found that changes in L1 levels do not affect the organization or expression of E-cadherin in cell lines, or in invading CRC tissue cells, and no changes in other epithelial or mesenchymal markers were detected after L1 transfection. The introduction of major EMT regulators (Slug and Twist) into CRC cell lines reduced the levels of E-cadherin and induced fibronectin and vimentin, but unlike L1, Slug and Twist expression was insufficient for conferring metastasis. In CRC cells L1 did not specifically cosegregate with CSC markers including CD133, CD44, and EpCAM. L1-mediated metastasis required NF-κB signaling in cells harboring either high or low levels of endogenous E-cadherin. The results suggest that L1-mediated metastasis of CRC cells does not require changes in EMT and CSC markers and operates by activating NF-κβ signaling.