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A Bender

Publications and source records attributed to A Bender.

At least 73 records · Page 4Linked to original sources
PubMed1993

Genetic evidence for the roles of the bud-site-selection genes BUD5 and BUD2 in control of the Rsr1p (Bud1p) GTPase in yeast.

Yeast cells normally display either an axial (for MATa or MAT alpha cells) or bipolar (for MATa/alpha cells) pattern of bud-site selection. The RSR1 gene, which was previously identified as a multicopy suppressor of Ts- mutations in the bud-emergence gene CDC24, encodes a GTPase of the Ras family that is required for both budding patterns. Mutations in Rsr1p that presumably block its ability to bind or hydrolyze GTP cause a randomized budding phenotype, suggesting that regulators of Rsr1p will prove to be required for proper bud positioning. The BUD5 gene product is required for proper bud-site selection and contains similarity to GDP-dissociation stimulators (GDS) for Ras-type proteins, suggesting that Bud5p may be a GDS for Rsr1p. Here I report that BUD5 is required for wild-type RSR1, but not for mutationally activated rsr1val12, to serve as a multicopy suppressor of cdc24, indicating that Bud5p functions as a GDS for Rsr1p in vivo. To identify the GAP (GTPase-activating protein) for Rsr1p, a genetic selection was designed based on the observation that mutationally activated rsr1val12, but not wild-type RSR1, can serve as a multicopy suppressor of yeast RAS2(Ts) mutants. Mutants were selected that allowed wild-type RSR1 to act as a multicopy suppressor of RAS2(Ts). Two such mutations proved to be in the BUD2 gene, suggesting that Bud2p functions as a GAP for Rsr1p in vivo.

Cell Cycle Proteins↗
PubMed1993

The potentiating effect of LPS on tumor necrosis factor-alpha production by influenza A virus-infected macrophages.

Infection of murine PU5-1.8 macrophages and human monocytes by influenza A virus was associated with virus replication, release of tumor necrosis factor-alpha (TNF-alpha) and subsequent cell death. In the presence of small and by itself rather inefficient concentrations of lipopolysaccharide (LPS) or free lipid A (1 to 10 ng/ml), TNF-alpha production of virus-infected macrophages was strongly potentiated. LPS-triggered and enhanced TNF-alpha release from virus-infected macrophages was neither due to increased cell survival nor altered virus replication, potentiated TNF-alpha gene transcription, release of intracellularly stored TNF-alpha or shifts in the kinetics of TNF-alpha secretion. Influenza A virus infection alone induced a massive TNF-alpha mRNA accumulation which, however, was only weakly translated into bioactive TNF-alpha protein. When these virus-primed macrophages were exposed to LPS either simultaneously or up to 4 h after infection, an efficient and high translation into TNF-alpha protein occurred. Although the LPS-induced biochemical pathways leading to an augmented TNF-alpha production by virus-infected macrophages still remains unsolved, the findings suggest that the frequently observed serious clinical complications in the course of combined influenza A virus and bacterial infections may be due, at least in part, to an excessive release of cytokines such as TNF-alpha.

Animals↗
PubMed1993

Role of macrophage cytokines in influenza A virus infections.

Human monocytes and murine macrophages were found to be susceptible to infection by influenza A virus. Although virus replication was low, infection led to cell death which was characterized by an extreme intracellular vacuolization. Most importantly, influenza A virus infection was accompanied by a particular pattern of cytokine release. Whereas IL-1 beta, IL-6 and TNF-alpha production was dependent on exposure to infectious virus, IFN-alpha/beta release was also induced by UV-inactivated virus. Although influenza A virus infection alone induced a substantial cytokine mRNA accumulation, translation into bioactive cytokine protein was rather limited. However, addition of low LPS concentrations was capable of strongly potentiating cytokine release from virus-infected cells. Thus, in a first step, an influenza A virus infection primes mononuclear phagocytes by leading to an accumulation of cytokine mRNA which, in a second step, may be readily translated into bioactive cytokines when triggering signals such as LPS are available. These findings suggest that influenza A virus represents an ultimately fatal macrophage activating factor which, when inducing moderate amounts of cytokines, may be beneficial by mounting an immediate antiviral response, but which may cause adverse effects when cytokine release is highly elevated by bacterial products.

Animals↗
PubMed1993

Clonal specificity of human gamma delta T cells: V gamma 9+ T-cell clones frequently recognize Plasmodium falciparum merozoites, Mycobacterium tuberculosis, and group-A streptococci.

Peripheral blood gamma delta T cells expressing a V gamma 9/V delta 2 T-cell receptor are stimulated by killed bacteria including Mycobacterium tuberculosis (m.tb.) and group-A streptococci (strep A). In addition, recent data indicate that V gamma 9/V delta 2 T cells from unexposed individuals also respond to Plasmodium falciparum (P. falcip.) merozoites. Here we analyzed the reactivity to these ligands of 23 V gamma 9/V delta 2, 3 V gamma 9/V delta 1, and 4 V gamma 9-/V delta 1 clones derived from 8 healthy individuals after phytohemagglutinin stimulation of cell sorter-selected gamma delta T cells. Upon restimulation in the presence of irradiated antigen-presenting cells, the majority of V gamma 9/V delta 2 clones recognized m.tb. and strep A (but not strep D), and about one third of the clones also recognized P. falcip. Some clones, however, recognized only one or two of the tested ligands, and 4 V gamma 9/V delta 2 clones did not react at all. Interestingly, 2 of 3 V gamma 9/V delta 1 clones proliferated in response to m.tb., P. falcip., strep A and strep D, while V gamma 9-/V delta 1 clones were not activated by any of the tested ligands. Nucleotide sequence analysis indicated a broad diversity of V gamma 9 N regions in V gamma 9/V delta 2 clones. At the clonal level, our results demonstrate that individual V gamma 9/V delta 2 T cells can recognize m.tb., strep A, as well as P. falcip.-infected erythrocytes, with no influence of the expressed V gamma 9 N region.

Animals↗
PubMed1992

A yeast gene (BEM1) necessary for cell polarization whose product contains two SH3 domains.

Cell polarization requires that a cellular axis or cell-surface site be chosen and that the cytoskeleton be organized with respect to it. Details of the link between the cytoskeleton and the chosen axis or site are not clear. Cells of the yeast Saccharomyces cerevisiae exhibit cell polarization in two phases of their life cycle, during vegetative growth and during mating, which reflects responses to intracellular and extracellular signals, respectively. Here we describe the isolation of two mutants defective specifically in cell polarization in response to peptide mating pheromones. The mutants carry special alleles (denoted bem1-s) of the BEM1 gene required for cell polarization during vegetative growth. Unlike other bem1 mutants, the bem1-s mutants are normal for vegetative growth. Complete deletion of BEM1 leads to the defect in polarization of vegetative cells seen in bem1 mutants. The predicted sequence of the BEM1 protein (Bem1p) reveals two copies of a domain (denoted SH3) that is found in many proteins associated with the cortical cytoskeleton and which may mediate binding to actin or some other component of the cell cortex. The sequence of Bem1p and the properties of mutants defective in this protein indicate that it may link the cytoskeleton to morphogenetic determinants on the cell surface.

Adaptor Proteins, Signal Transducing↗
PubMed1992

A Ser/Thr-rich multicopy suppressor of a cdc24 bud emergence defect.

MSB2 was identified previously as a multicopy suppressor of a temperature-sensitive mutation in CDC24, a gene required for polarity establishment and bud formation in Saccharomyces cerevisiae. The inferred MSB2 product contains 1306 amino acids, 42% of which are Ser or Thr. Its Ser+Thr-richness and hydrophobicity profile suggest that Msb2p may be an integral membrane protein containing a long, periplasmic, N-terminal domain and a short, cytoplasmic, C-terminal domain. Cells that lack MSB2 display no obvious mutant phenotypes. MSB2 is located between the centromere and KSS1 on the right arm of chromosome VII. Although physical mapping suggests that MSB2 and LEU1 (on the left arm of chromosome VII) are approximately 40 kb apart, the genetic map distance observed between leu1 and an msb2::URA3 marker was only 2.3 cM.

Amino Acid Sequence↗
PubMed1992

Preferential activation of peripheral blood V gamma 9+ gamma/delta T cells by group A, B and C but not group D or F streptococci.

Previous studies have established that inactivated mycobacteria are potent and selective activators of V gamma 9+/V delta 2+ human gamma/delta T cells. Here we have analysed the proliferative response of human gamma/delta T cells to five serologically distinct groups of streptococci. While heat-inactivated streptococci of all five serogroups tested (A, B, C, D and F) induced a strong proliferative response in peripheral blood mononuclear cells (PBMC), only groups A, B and C elicited a selective activation of V gamma 9+ gamma/delta T cells in 10 (serogroup B) or 11 (serogroups A and C) of 11 tested healthy individuals. In striking contrast, groups D and F streptococci failed to activate gamma/delta T cells in nine of 11 donors and induced only a weak gamma/delta T cell response in two additional individuals. Depletion of V gamma 9+ T cells before culture completely eliminated all gamma/delta T cell responses to streptococci. These data indicate that groups A, B and C (but not D or F) streptococci can be included in the growing list of selective ligands for V gamma 9+/V delta 2+ human gamma/delta T cells.

Cells, Cultured↗
PubMed1992

RSR1, a ras-like gene homologous to Krev-1 (smg21A/rap1A): role in the development of cell polarity and interactions with the Ras pathway in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae ras-like gene RSR1 is particularly closely related to the mammalian gene Krev-1 (also known as smg21A and rap1A). RSR1 was originally isolated as a multicopy suppressor of a cdc24 mutation, which causes an inability to bud or establish cell polarity. Deletion of RSR1 itself does not affect growth but causes a randomization of bud position. We have now constructed mutant alleles of RSR1 encoding proteins with substitutions of Val for Gly at position 12 (analogous to constitutively activated Ras proteins) or Asn for Lys at position 16 (analogous to a dominant-negative Ras protein). rsr1Val-12 could not restore a normal budding pattern to an rsr1 deletion strain but could suppress a cdc24 mutation when overexpressed. rsr1Asn-16 could randomize the budding pattern of a wild-type strain even in low copy number but was not lethal even in high copy number. These and other results suggest that Rsr1p functions only in bud site selection and not in subsequent events of polarity establishment and bud formation, that this function involves a cycling between GTP-bound and GDP-bound forms of the protein, and that the suppression of cdc24 involves direct interaction between Rsr1p[GTP] and Cdc24p. Functional homology between Rsr1p and Krev-1 p21 was suggested by the observations that expression of the latter protein in yeast cells could both suppress a cdc24 mutation and randomize the budding pattern of wild-type cells. As Krev-1 overexpression can suppress ras-induced transformation of mammalian cells, we looked for effects of RSR1 on the S. cerevisiae Ras pathway. Although no suppression of the activated RAS2Val-19 allele was observed, overexpression of rsr1Val-12 suppressed the lethality of strains lacking RAS gene function, apparently through a direct activation of adenyl cyclase. This interaction of Rsr1p with the effector of Ras in S. cerevisiae suggests that Krev-1 may revert ras-induced transformation of mammalian cells by affecting the interaction of ras p21 with its effector.

Adenylyl Cyclases↗
PubMed1991

Influenza A virus infection of macrophages. Enhanced tumor necrosis factor-alpha (TNF-alpha) gene expression and lipopolysaccharide-triggered TNF-alpha release.

We have previously shown that infection of macrophages by influenza A virus is capable of priming for a high TNF-alpha production in response to LPS. The present study was designed to examine in more detail TNF-alpha gene expression and TNF-alpha protein release of virus-infected, murine PU5-1.8 macrophages in the presence or absence of low and by itself rather inefficient concentrations of LPS (10 ng/ml). Although influenza A virus infection alone induced a massive TNF-alpha mRNA accumulation, translation into the bioactive TNF-alpha protein was low as intra- and extracellularly determined by bioassay, specific ELISA and Western blot. However, when LPS was added simultaneously or up to 4 h after infection, a high TNF-alpha production was initiated. The virus-induced TNF-alpha mRNA accumulation appeared to be due to both transcriptional and post-transcriptional changes: an enhanced TNF-alpha gene transcription as determined by nuclear run-on transcription assay and a markedly prolonged half-life of TNF-alpha mRNA as shown in actinomycin D-treated macrophages. These findings imply that influenza A virus may 1) either directly or indirectly stimulate TNF-alpha gene transcription activators or may interfere with labile transcription repressor proteins and 2) may stabilize TNF-alpha mRNA by delaying its degradation. Both mechanisms, taken together, prime influenza A virus-infected macrophages for a high TNF-alpha release in response to LPS which, as clinical cases show, may adversely affect patients with combined influenza A virus and bacterial infections.

Animals↗
PubMed1991

The primary response of human gamma/delta + T cells to Mycobacterium tuberculosis is restricted to V gamma 9-bearing cells.

We have previously reported that peripheral blood gamma/delta + T cells proliferate in high frequency (1 in 2-20) in response to heat-killed Mycobacterium tuberculosis (M.tb.). In the present study, the T cell receptor phenotype of mycobacteria-responsive human gamma/delta + T cells was analyzed in primary cultures with a set of monoclonal antibodies (mAbs) directed against V gamma 9, V delta 1, and V delta 2. When unseparated T cells were stimulated with M.tb., all proliferating gamma/delta + T cells expressed V gamma 9 (and V delta 2) after culture. Selective depletion of V gamma 9-bearing cells before culture completely abolished the proliferative response of all gamma/delta + cells (but did not inhibit reactivity of alpha/beta + T cells). In addition, when CD4- CD8- thymocytes were stimulated with M.tb., there was again selective outgrowth of V gamma 9+ cells. In this case, the starting responder population contained few (0.5-1.8%) V gamma 9+ and many (11.5-31.5%) V delta 1+ cells that did not coexpress V gamma 9. These V delta 1+ cells were not activated by M.tb. but could be readily stimulated by anti-V delta 1 mAb A13. Finally, a V gamma 9-specific mAb selectively suppressed the proliferative response of gamma/delta + T cells to M.tb. Taken together, our results demonstrate that, within gamma/delta + T cells, reactivity towards M.tb. is an exclusive property of V gamma 9+/V delta (2+)-bearing cells.

Antibodies, Monoclonal↗
PubMed1991

T cell receptor/CD3-signaling induces death by apoptosis in human T cell receptor gamma delta + T cells.

mAb directed against the TCR/CD3 complex activate resting T cells. However, TCR/CD3 signaling induces death by apoptosis in immature (CD4+CD8+) murine thymocytes and certain transformed leukemic T cell lines. Here we show that anti-TCR and anti-CD3 mAb induce growth arrest of cloned TCR-gamma delta + T cells in the presence of IL-2. In the absence of exogenous IL-2, however, the very same anti-TCR/CD3 mAb stimulated gamma delta (+)-clones to proliferation and IL-2 production. In the presence of exogenous IL-2, anti-TCR/CD3 mAb induced the degradation of DNA into oligosomal bands of approximately 200 bp length in cloned gamma delta + T cells. This pattern of DNA fragmentation is characteristic for the programmed cell death termed apoptosis. These results demonstrate that TCR/CD3 signaling can induce cell death in cloned gamma delta + T cells. In addition, this report is the first to show that apoptosis triggered by TCR/CD3 signaling is not restricted to CD4+CD8+ immature thymocytes and transformed leukemic T cell lines but can be also observed with IL-2-dependent normal (i.e., TCR-gamma delta +) T cells.

Antibodies, Monoclonal↗
PubMed1991

Activation and activation-driven death of human gamma/delta T cells.

We have concentrated on three functional aspects of human gamma/delta+ T cells. (1) Peripheral blood gamma/delta+ T cells proliferate at a high frequency in response to heat-killed mycobacteria (M.tb.). We present evidence that the primary response of human gamma/delta+ T cells is restricted to those cells bearing a V gamma 9/V delta 2 receptor. (2) T cells expressing the alpha/beta TCR can be activated through the CD2 antigen via an "alternative" pathway. Activation requires the combined signaling of 2 mAb directed against two distinct epitopes of CD2. In striking contrast, cloned gamma/delta+ T cells are activated by a single immobilized anti-CD2 mAb directed against the sheep E-binding epitope of CD2. Stimulation of cloned gamma/delta+ T cells by a single anti-CD2 mAb results in IL-2 production, proliferation, and triggering of cytolytic effector function. (3) Cloned gamma/delta+ T cells are susceptible to programmed cell death (apoptosis) when stimulated simultaneously by anti-CD3/TCR mAb plus exogenous IL-2. The very same clones are activated, however, by the same stimuli in the absence of exogenous IL-2 (but in the presence of LCL feeder cells). This provides the basis for a model where extensive gamma/delta+ T cell proliferation is negatively regulated by antigen plus IL-2.

Antibodies, Monoclonal↗
PubMed1991

Use of a screen for synthetic lethal and multicopy suppressee mutants to identify two new genes involved in morphogenesis in Saccharomyces cerevisiae.

Genes CDC24 and CDC42 are required for the establishment of cell polarity and for bud formation in Saccharomyces cerevisiae. Temperature-sensitive (Ts-) mutations in either of these genes cause arrest as large, unbudded cells in which the nuclear cycle continues. MSB1 was identified previously as a multicopy suppressor of Ts- cdc24 and cdc42 mutations. We have now sequenced MSB1 and constructed a deletion of this gene. The predicted amino acid sequence does not closely resemble any other in the available data bases, and the deletion does not produce any readily detectable phenotype. However, we have used a colony-sectoring assay to identify additional genes that appear to interact with MSB1 and play a role in bud emergence. Starting with a strain deleted for the chromosomal copy of MSB1 but containing MSB1 on a high-copy-number plasmid, mutants were identified in which MSB1 had become essential for viability. The new mutations defined two genes, BEM1 and BEM2; both the bem1 and bem2 mutations are temperature sensitive and are only partially suppressed by MSB1. In bem1 cells, a single copy of MSB1 is necessary and sufficient for viability at 23 or 30 degrees C, but even multiple copies of MSB1 do not fully suppress the growth defect at 37 degrees C. In bem2 cells, a single copy of MSB1 is necessary and sufficient for viability at 23 degrees C, multiple copies are necessary for viability at 30 degrees C, and even multiple copies of MSB1 do not suppress the growth defect at 37 degrees C. In a wild-type background (i.e., a single chromosomal copy of MSB1), both bem1 and bem2 mutations cause cells to become large and multinucleate even during growth at 23 degrees C, suggesting that these genes are involved in bud emergence. This suggestion is supported for BEM1 by other evidence obtained in a parallel study (J. Chant, K. Corrado, J. Pringle, and I. Herskowitz, submitted for publication). BEM1 maps centromere distal to TYR1 on chromosome II, and BEM2 maps between SPT15 and STP2 on chromosome V.

Amino Acid Sequence↗
PubMed1991

Influenza A virus infects macrophages and stimulates release of tumor necrosis factor-alpha.

The clinical picture of influenza A virus infections indicates that release of tumor necrosis factor-alpha (TNF-alpha) may be involved. In the present study we exposed the murine macrophage line PU5-1.8 to influenza A virus and observed a productive infection which was followed by subsequent cell death. Infection of macrophages was accompanied by TNF-alpha mRNA accumulation and TNF-alpha release. TNF-alpha production could only be induced by live virus whereas interferon release was also stimulated by inactivated virus. When virus-infected macrophages were exposed to low amounts of lipopolysaccharide (LPS; 1-10 ng/ml) TNF-alpha production was strongly potentiated. These data show that low LPS concentrations could readily trigger a high TNF-alpha release from influenza-A-virus-infected macrophages which could, at least partially, explain the serious complications of combined influenza A virus and bacterial infections.

Animals↗
PubMed1990

Human cytotoxic lymphocytes. V. Frequency and specificity of gamma delta+ cytotoxic lymphocyte precursors activated by allogeneic or autologous stimulator cells.

We have investigated the frequency and specificity of gamma delta+ cytotoxic lymphocyte precursors (CLP) under limiting dilution culture conditions. E rosette separated total T cells and CD3+CD4-CD8-TCR alpha beta- double-negative (DN) T cells were cocultured with allogeneic or autologous PBMC stimulator cells, and frequencies of alloreactive and autoreactive CLP were determined after 12 to 14 days against Con A blast target cells. Freshly isolated DN cells consisting of 82.3 +/- 8.2% gamma delta+ T cells did not exert cytolytic activity against K562 or anti-TCR gamma delta mAb-producing hybridoma cells. In striking contrast to E+ cells, the vast majority of alloantigen-stimulated clonally developing DN CLP did not show specificity for stimulator-derived target cells. Thus, frequencies of alloreactive and autoreactive CLP after alloantigenic stimulation were in the range of 1/100 to 1/4800 and 1/450 to 1/5000, respectively. After coculture with autologous stimulator cells, frequencies of autoreactive and alloreactive DN CLP were 1/700 to 1/2700 and 1/1360 to 1/4500, respectively. Split culture analysis revealed that most proliferating DN colonies selected for high probability of clonality simultaneously killed both autologous and HLA-mismatched allogeneic targets. The majority of the DN cells expressed the CD3+/TCR gamma delta+ phenotype after culture, and thus were not CD2+CD3- NK cells. Taken together, our results show that 1) freshly isolated peripheral blood gamma delta+ T cells lack cytotoxic activity, and 2) most cytotoxic gamma delta+ T cells activated by autologous or allogeneic stimulator cells under limiting dilution conditions do not discriminate between autologous and allogeneic targets.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1990

Tumor necrosis factor-alpha production of influenza A virus-infected macrophages and potentiating effect of lipopolysaccharides.

Influenza A virus infections are commonly associated with symptoms that suggest involvement of TNF-alpha. In this study, we exposed human monocytes, rat alveolar macrophages, and murine PU5-1.8 macrophages to influenza A virus, strain Puerto Rico 8. We observed a productive infection that was accompanied by TNF-alpha mRNA accumulation, TNF-alpha release and subsequent cell death. TNF-alpha production was dependent on exposure to live virus, in contrast to IFN release that was also induced by UV-inactivated virus. Most strikingly, low amounts of LPS (1 to 10 ng/ml) from Escherichia coli or Haemophilus influenzae were capable of strongly potentiating TNF-alpha production from virus-infected macrophages. The potentiating effect of LPS was neither due to increased survival of macrophages nor to altered virus multiplication, enhanced TNF-alpha gene expression, discharge of intracellular TNF-alpha stores, or shifts in the kinetics of TNF-alpha release. Thus, low amounts of LPS, which could easily be present in vivo, may serve as a potent trigger signal for TNF-alpha production from macrophages that have been primed by influenza A virus infection. These data suggest that the frequently observed serious complications of combined influenza A virus and bacterial infections may be partially due to a high TNF-alpha production.

Blotting, Northern↗
PubMed1990

A large fraction of human peripheral blood gamma/delta + T cells is activated by Mycobacterium tuberculosis but not by its 65-kD heat shock protein.

We report that M. tuberculosis organisms, but neither PHA nor allogeneic stimulator cells, preferentially activate gamma/delta+ cells within E rosette-purified peripheral blood T cells. gamma/delta+ T cells from purified protein derivative (PPD)-nonimmune healthy donors were enriched by depletion of CD4+ and CD8+ cells; double-negative (DN) cells contained 65-92% gamma/delta+ T cells. Limiting dilution (LD) analyses revealed that 1 of 2-19 purified DN cells proliferated in response to mycobacteria, while frequencies of DN cells proliferating in response to a recombinant 65-kD heat shock protein (hsp 65) of M. tuberculosis/M. bovis were 10-20-fold lower. Established clones of mycobacteria-reactive gamma/delta+ T cells specifically recognized mycobacteria, but neither PPD nor hsp 65. Restimulation of these clones required the presence of PBMC feeder cells; EBV-transformed lymphoblastoid cell lines could not substitute for PBMC. Mycobacteria-reactive gamma/delta+ clones proliferated equally well in the presence of autologous or allogeneic (HLA-DR-different) PBMC feeder cells and thus were not MHC class II restricted. Taken together, these results demonstrate that mycobacteria-reactive gamma/delta+ T cells are present in high frequency in the peripheral blood of healthy individuals, and suggest that hsp 65 of mycobacteria is not a major antigen for gamma/delta+ T cells of normal PPD-nonimmune blood donors.

HLA-DR Antigens↗