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At least 1,315 records · Page 73Linked to original sources
PubMed2006

Arenavirus Z protein as an antiviral target: virus inactivation and protein oligomerization by zinc finger-reactive compounds.

Several disulfide-based and azoic compounds have shown antiviral and virucidal properties against arenaviruses in virus yield-inhibition and inactivation assays, respectively. The most effective virucidal agent, the aromatic disulfide NSC20625, was able to inactivate two strains of the prototype arenavirus species Lymphocytic choriomeningitis virus (LCMV). Inactivated viral particles retained the biological functions of the virion envelope glycoproteins in virus binding and uptake, but were unable to perform viral RNA replication. Furthermore, in inactivated virions, the electrophoretic profile of the Z protein was altered when analysed under non-reducing conditions, whereas the patterns of the proteins NP and GP1 remained unaffected. Treatment of a recombinant LCMV Z protein with the virucidal agents induced unfolding and oligomerization of Z to high-molecular-mass aggregates, probably due to metal-ion ejection and the formation of intermolecular disulfide bonds through the cysteine residues of the Z RING finger. NSC20625 also exhibited antiviral properties in LCMV-infected cells without affecting other cellular RING-motif proteins, such as the promyelocytic leukaemia protein PML. Altogether, the investigations described here illustrate the potential of the Z protein as a promising target for therapy and the prospects of the Z-reactive compounds to prevent arenavirus dissemination.

Animals↗
PubMed1999

Multiple developmental roles for CRAC, a cytosolic regulator of adenylyl cyclase.

Receptor-mediated activation of adenylyl cyclase (ACA) in Dictyostelium requires CRAC protein. Upon translocation to the membrane, this pleckstrin homology (PH) domain protein stimulates ACA and thereby mediates developmental aggregation. CRAC may also have roles later in development since CRAC-null cells can respond to chemotactic signals and participate in developmental aggregation when admixed with wild-type cells, but they do not complete development within such chimeras. To test whether the role of CRAC in postaggregative development is related to the activation of ACA, chemotactic aggregation was bypassed in CRAC-null cells by activating the cAMP-dependent protein kinase (PKA). While such strains formed mounds, they did not complete fruiting body morphogenesis or form spores. Expression of CRAC in the prespore cells of these strains rescued sporulation and fruiting body formation. This later function of CRAC does not appear to require its PH domain since the C-terminal portion of the protein (CRAC-DeltaPH) can substitute for full-length CRAC in promoting spore cell formation and morphogenesis. No detectable ACA activation was observed in any of the CRAC-null strains rescued by PKA activation and expression of CRAC-DeltaPH. Finally, we found that the development of CRAC-null ACA-null double mutants could be rescued by the activation of PKA together with the expression of CRAC-DeltaPH. Thus, there appears to be a required function for CRAC in postaggregative development that is independent of its previously described function in the ACA activation pathway.

Adenylyl Cyclases↗
PubMed1997

The dorsalizing and neural inducing gene follistatin is an antagonist of BMP-4.

Specific signaling molecules play a pivotal role in the induction and specification of tissues during early vertebrate embryogenesis. BMP-4 specifies ventral mesoderm differentiation and inhibits neural induction in Xenopus, whereas three molecules secreted from the organizer, noggin, follistatin and chordin dorsalize mesoderm and promote neural induction. Here we report that follistatin antagonizes the activities of BMP-4 in frog embryos and mouse teratocarcinoma cells. In Xenopus embryos follistatin blocks the ventralizing effect of BMP-4. In mouse P19 cells follistatin promotes neural differentiation. BMP-4 antagonizes the action of follistatin and prevents neural differentiation. In addition we show that the follistatin and BMP-4 proteins can interact directly in vitro. These data provide evidence that follistatin might play a role in modulating BMP-4 activity in vivo.

Animals↗
PubMed2002

Multiple signalling pathways connect chemoattractant receptors and calcium channels in Dictyostelium.

Dictyostelium mutants expressing aequorin were used to study and compare the roles of heterotrimeric G-proteins and the second messengers IP3 and cGMP in regulating folate- and cAMP receptor-activated [Ca2+]i signals. The calcium responses of vegetative cells to folate were dramatically impaired in Gbeta and Galpha4 null mutants but were restored with altered kinetics and temperature-sensitivity in Gbeta null mutants overexpressing wild type and temperature-sensitive Gbeta isoforms. Folic acid receptors thus mediate changes in [Ca2+]i via a Galpha4betagamma-dependent pathway. Neither folate nor cAMP-induced [Ca2+]i signals were significantly altered in PLC null transformants, but [Ca2+]i changes elicited by both attractants were significantly prolonged in two stmF mutants lacking cGMP-specific phosphodiesterase activity. This confirms an important role of cGMP in regulating receptor-activated Ca2+ uptake and/or extrusion systems. This cGMP-dependent part of the Ca2+ response to cAMP stimuli was developmentally down-regulated and all but disappeared by the time the cells reached full aggregation competence after 8 h of starvation. The results suggest that folate and cAMP receptor-activated [Ca2+]i signals are regulated in a complex manner via multiple signalling pathways, one that is G-protein- and cGMP-dependent (present at the vegetative and early poststarvation stage) and another that is G-protein-independent (dominant in fully aggregation-competent cells at approximately 8 h poststarvation).

Animals↗
PubMed1990

Graded changes in dose of a Xenopus activin A homologue elicit stepwise transitions in embryonic cell fate.

The protein XTC-MIF, a Xenopus homologue of activin A and a potent mesoderm-inducing factor, can induce responding animal pole explants to form several different cell types in a dose-dependent manner, higher doses eliciting more dorso-anterior tissues. This graded response, characteristic of classically postulated morphogens, may underlie pattern formation, but the response of intact animal caps to XTC-MIF provides only a crude indication of trends. Here we report the effects of XTC-MIF on dispersed blastomeres rather than intact animal caps. Under these conditions, responding cells distinguish sharply between doses of pure XTC-MIF differing by less than 1.5-fold. Two different response thresholds have been found, defining three cell states. This suggests that XTC-MIF has an instructive effect. Notochord and muscle are both induced in the same narrow dose-range. Mixing treated with untreated cells does not seem to shift the dose thresholds, showing that at least some cells can stably record the received dose of inducing factor.

Actins↗
PubMed2003

Functional and molecular characterization of the epithelioid to round transition in human colorectal cancer LoVo cells.

In subclones of the human colon cancer LoVo cell line, there is a reproducible spontaneous transition from an epithelioid (E) to a round (R) morphotype. The E to R transition is associated with increased cell growth, absence of E-cadherin-dependent compaction in a slow aggregation assay, loss of contact inhibition of motility and directional migration in a wound filling motility assay. Furthermore, none of the E subclones from LoVo was invasive into chick heart fragments. This is in contrast to the R subclones that were either nonadherent or adherent and invasive. Macroarray analysis demonstrated transcriptional downregulation of plakoglobin in R type LoVo cells and this was confirmed at the level of the mRNA by quantitative RT-PCR. Western blotting showed lower expression of all components of the E-cadherin/catenin complex in R subclones. Interestingly, treatment of R subclones with the demethylating agent 5-aza-2'-deoxycytidine resulted in restoration of the E morphotype, higher expression of E-cadherin, but not plakoglobin mRNA, and higher expression of E-cadherin and plakoglobin at the protein level.

Azacitidine↗
PubMed2002

krox-20/egr-2 is up-regulated following non-specific and homophilic adhesion in rat macrophages.

Macrophages are known to adhere to a plastic dish via beta2 integrin (CR3) and scavenger receptors. Although their functions such as phagocytosis, endocytosis, and nitric oxide production have been investigated on adherent macrophages in vitro, very little is known about intracellular signals triggered by adhesion to a plastic dish. Recently we reported that the mRNA level of krox-20/egr-2 was significantly increased in rat alveolar macrophages following exposure to fibrous titanium dioxide particles. In the present study we report that up-regulation of krox-20/egr-2 gene expression following adhesion to a plastic dish and homophilic adhesion in rat alveolar macrophages and rat macrophage cell line, NR8383. The mRNA level of krox-20/egr-2 increased with a peak 1 hr after adhesion to a plastic dish in both cell types. Piceatannol inhibited tyrosine-phosphorylation of Syk and decreased both adhesion and krox-20/egr-2 mRNA level. In contrast staurosporine, a serine/threonine kinase inhibitor, increased adherence of macrophages and yet prohibited the adhesion-dependent increase in krox-20/egr-2 gene expression. When NR8383 cells are cultured in suspension, the cells aggregated naturally and produced cell clumps. The mRNA level of krox-20/egr-2 also increased in response to the homophilic intercellular adhesion. The increased mRNA level of krox-20/egr-2 was not caused by inflammatory stimuli, because lipopolysaccharide did not affect the aggregation-dependent up-regulation of krox-20/egr-2 gene. The up-regulation of krox-20/egr-2 gene due to the homophilic cell aggregation was also inhibited either by piceatannol or staurosporine. Those results suggest that krox-20/egr-2 gene expression is triggered by sensing non-specific and homophilic cellular adhesion and the following phosphorylation of signal transducing proteins including Syk and staurosporine-inhibitable kinases.

Animals↗
PubMed1983

Specific cell-cell contact serves as the developmental signal to deactivate discoidin I gene expression in Dictyostelium discoideum.

Specific cell-cell contact is a major regulatory signal controlling cell differentiation in Dictyostelium discoideum, causing dramatic changes in the developmental program of gene expression. In this report, we focus on the relationships between specific cell-cell contact and the activity of the genes for discoidin I, an endogenous lectin that has been implicated in the cell-cell cohesion process. By performing quantitative RNA dot-hybridization assays and RNA gel blot-hybridization analyses, using as a probe a recombinant plasmid containing a discoidin I cDNA insert, we have measured changes in discoiding I mRNA levels during normal development and in response to specific manipulations of the state of cellular aggregation. Our major findings are as follows. (i) During normal development on filters, there is a close temporal correspondence between the establishment of specific cell-cell contacts and the decline in discoidin I mRNA levels. By the tight-aggregate stage, discoidin I mRNA is barely detectable. (ii) When tight aggregates are disaggregated and the cells are maintained in the disaggregated state, there is a dramatic rise in discoidin I mRNA content. (iii) When cells are developed in suspension (conditions that interfere with the establishment of tight cell-cell contacts), discoidin I mRNA accumulates to abnormally high levels, and these persist well after the levels in filter-developed cells have declined. Taken together, these results strongly suggest that cell-cell contact is the normal developmental signal to deactivate discoidin I gene expression; thus, a contact-deactivated gene for which a recombinant DNA probe is available has now been identified. Furthermore, we demonstrate that exogenous cAMP almost completely blocks the disaggregation-induced reactivation of discoidin I gene expression. Possible mechanistic relationships between specific cell-cell contact, intracellular cAMP levels, and developmental gene expression are discussed.

Journal Article↗
PubMed1989

Two-phase regulatory pathway controls cAMP receptor-mediated expression of early genes in Dictyostelium.

Two classes of early genes in Dictyostelium are differentially regulated by extracellular pulses of cAMP interacting with its cell-surface receptor, conditions that also regulate chemotaxis and aggregation. The pulse-repressed genes, such as K5, are induced shortly after the onset of starvation and are repressed a few hr later during aggregation by cAMP pulses. The pulse-induced genes (including D2, M3, and those encoding contact sites A, the G alpha protein subunit G alpha 2, and the cell-surface cAMP receptor) are maximally induced just prior to aggregation by pulses of cAMP and are subsequently repressed by sustained moderate levels of cAMP--conditions that exist sequentially in development. In this manuscript, we further analyze the requirement for cAMP pulses and characterize a requirement for protein synthesis for the expression of these two classes of genes. Our results indicate that the control of expression of both the pulse-induced and pulse-repressed genes requires other developmentally regulated factors in addition to starvation and cAMP pulses. We also identified another early gene, F9, whose expression is stimulated upon starvation, is not responsive to cAMP, and is hyperstimulated by cycloheximide, in a manner similar to the cycloheximide stimulation of c-fos and other serum-induced genes in mammalian cells. Examination of the kinetics of expression of the pulse-induced genes in a mutant blocked in the cAMP relay pathway indicates that their expression is controlled by a two-phase process. The first phase requires starvation and CMF, an extracellular conditioned medium factor, and results in a low level of expression. The second phase requires establishment of the cAMP signal-relay system and induces the genes to a high level. Both phases require prior and concomitant protein synthesis. Some of the members of the pulse-induced class encode elements of the cAMP signal-relay system that controls aggregation, indicating a feedback autoregulation. The two-phase process might allow the "finetuning" of the level of expression of genes involved in aggregation.

Adenylyl Cyclases↗
PubMed1997

Intercellular adhesion molecule-1 (ICAM-1) is expressed on human neutrophils and is essential for neutrophil adherence and aggregation.

This study investigated the expression and regulation of intercellular adhesion molecule-1 (ICAM-1) on human polymorphonuclear neutrophils (PMNs), and its potential role in PMN-PMN adherence and aggregation as observed during systemic inflammatory response syndrome. Normal human PMNs were found to express ICAM-1 with 90% positive population, and this expression was augmented by endotoxin (lipopolysaccharide, LPS) and tumor necrosis factor-alpha (TNF-alpha) stimulation. The presence of ICAM-1 mRNA in human PMNs was further detected by reverse transcription-polymerase chain reaction before and after LPS and TNF-alpha treatment. Furthermore, incubation of PMNs with LPS and TNF-alpha resulted in significant increases in PMN-PMN adherence and aggregation, while addition of either anti ICAM-1 mAb or anti CD11b/CD18 mAb significantly inhibited LPS and TNF-alpha-mediated PMN-PMN adherence and aggregation. These novel findings demonstrate that ICAM-1 is expressed on human PMNs and responsible for PMN aggregation, and suggest that the interaction between ICAM-1 and CD11b/CD18 may be the molecular basis for PMN aggregation and clumping in the microcirculation during systemic inflammatory response syndrome.

Adult↗
PubMed1999

A cell-counting factor regulating structure size in Dictyostelium.

Developing Dictyostelium cells form large aggregation streams that break up into groups of 0.2 x 10(5) to 1 x 10(5) cells. Each group then becomes a fruiting body. smlA cells oversecrete an unknown factor that causes aggregation streams to break up into groups of approximately 5 x 10(3) cells and thus form very small fruiting bodies. We have purified the counting factor and find that it behaves as a complex of polypeptides with an effective molecular mass of 450 kD. One of the polypeptides is a 40-kD hydrophilic protein we have named counting. In transformants with a disrupted counting gene, there is no detectable secretion of counting factor, and the aggregation streams do not break up, resulting in huge (up to 2 x 10(5) cell) fruiting bodies.

Amino Acid Sequence↗
PubMed1991

Non-dimer DNA damage in Chinese hamster V-79 cells exposed to ultraviolet-B light.

To understand and characterize non-dimer DNA damage and cytotoxicity induced by ultraviolet-B light (UV-B, 290-320 nm), an alkaline elution technique for analysis of DNA damage was used on Chinese hamster V-79 cells. Ultraviolet-B exposure produced a dose-dependent induction of DNA single strand breaks and DNA-protein crosslinks; however, there was an absence of DNA-DNA interstrand crosslinks. Neither of these types of DNA damage were repaired within a a 24 h incubation of the cells following a single UV-B exposure; rather the damage increased. Using a colony forming assay, we found that UV-B exposure resulted in an increase of cytotoxicity in a dose-dependent fashion. In addition, UV-B exposure inhibited DNA and RNA synthesis. The role of non-dimer DNA damage in the cytotoxicity induced by UV-B is discussed.

Animals↗
PubMed2005

Phosphorylation of bluetongue virus nonstructural protein 2 is essential for formation of viral inclusion bodies.

In bluetongue virus (BTV)-infected cells, large cytoplasmic aggregates are formed, termed viral inclusion bodies (VIBs), which are believed to be the sites of viral replication and morphogenesis. The BTV nonstructural protein NS2 is the major component of VIBs. NS2 undergoes intracellular phosphorylation and possesses a strong single-stranded RNA binding activity. By changing phosphorylated amino acids to alanines and aspartates, we have mapped the phosphorylated sites of NS2 to two serine residues at positions 249 and 259. Since both of these serines are within the context of protein kinase CK2 recognition signals, we have further examined if CK2 is involved in NS2 phosphorylation by both intracellular colocalization and an in vitro phosphorylation assay. In addition, we have utilized the NS2 mutants to determine the role of phosphorylation on NS2 activities. The data obtained demonstrate that NS2 phosphorylation is not necessary either for its RNA binding properties or for its ability to interact with the viral polymerase VP1. However, phosphorylated NS2 exhibited VIB formation while unmodified NS2 failed to assemble as VIBs although smaller oligomeric forms of NS2 were readily formed. Our data reveal that NS2 phosphorylation controls VIBs formation consistent with a model in which NS2 provides the matrix for viral assembly.

Amino Acid Sequence↗
PubMed1991

Isolation and characterization of human melanoma cell variants expressing high and low levels of CD44.

Variants of the human melanoma cell line LT5.1 were selected for high and low expression of the Mr 90,000 CD44 glycoprotein by using the Hermes-3 monoclonal antibody combined with fluorescence-activated cell sorting. Cells were single cell cloned and clones of CD44 high-expressing and CD44 low-expressing phenotype were isolated. The variants, which exhibited up to a 7-fold difference between high and low expression, have maintained a stable phenotype over a period of 3 months in tissue culture. Northern blot analysis of mRNA from the different clones showed correlation of levels of transcripts with fluorescence-activated cell sorting analysis data. Wound migration assays, utilizing the different clones, showed that the low-expressing clones manifested less motility than did cells showing high levels of CD44. Homotypic aggregation of cells was increased in those cells expressing high levels of CD44, and these variants were also better able to adhere to hyaluronate substrates. All of these activities were inhibited by the presence of anti-CD44 antibody. When injected i.v. into nu/nu BALB/c mice, the low-expressing clones gave significantly fewer lung nodules than the high-expressing clones, although the two variant types did not differ in their capacity to form s.c. tumors in similar mice. These results suggest that the CD44 molecule, possibly as a function of its activities as a hyaluronate receptor, may play a vital role in determining the fate of hematogenously disseminating melanoma cells.

Animals↗
PubMed1979

Isolation of cytopathogenic Rotavirus from neonatal calves.

Rotavirus was isolated in rolled calf kidney cultures from the intestinal contents of a calf suffering from diarrhoea. The cytopathic effect was demonstrated in native cultures as well as in stained preparations. The affected cells were elongated, became sickle-like in shape, disintegrated and detached from the monolayer. Virus-specific direct immunofluorescence ran parallel, in both time and intensity, with the cytopathic effects. Immuno-electron microscopy showed aggregated virus particles corresponding in size, shape and structure to Rotavirus virion.

Animals↗
PubMed1989

Control of mRNA stability during development of Dictyostelium discoideum.

A large group of mRNA species (which are mainly pre-spore specific) accumulate only after the formation of multicellular aggregates. They are transcribed at a constant rate from the beginning of development and their accumulation is controlled by a 10-20-fold increase in their stability. This mRNA stabilization is dependent upon multicellularity. When aggregates are dispersed, the mRNAs are destabilized; if cells are allowed to reaggregate, the destabilization is reversed. Destabilization is not due to a selective exclusion of mRNA from polyribosomes, but is a primary control event. It does not require synthesis of new RNA or protein, but it may require an interaction between ribosome and the 5'-end of mRNA molecules.

Cyclic AMP↗