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A Bender

Publications and source records attributed to A Bender.

At least 55 records · Page 3Linked to original sources
PubMed1996

Differential expression of perforin in muscle-infiltrating T cells in polymyositis and dermatomyositis.

Polymyositis (PM) and dermatomyositis (DM) are the prototypical inflammatory diseases of skeletal muscle. In PM, CD8+ T cells invade and destroy muscle fibers, whereas humoral effector mechanisms prevail in DM. We studied the expression of the cytotoxic mediator perforin in inflammatory cells in PM and DM muscle by semiquantitative PCR, immunohistochemistry and confocal laser microscopy. Similar levels of perforin mRNA were expressed in PM and DM, and abundant perforin-expressing CD3+CD8+ and CD3+ CD4+ T cells were observed in both diseases. However, there was a striking difference in the intracellular localization of perforin. In DM, perforin was distributed randomly in the cytoplasm of the inflammatory T cells. In contrast, 43% of the CD8+ T cells that contacted a muscle fiber in PM showed perforin located vectorially towards the target muscle fiber. The results suggest (a) that the random distribution of perforin in the cytoplasm of muscle-infiltrating T cells observed in DM reflects nonspecific activation, and (b) that the vectorial orientation observed only in PM reflects the specific recognition via the T cell receptor of an antigen on the muscle fiber surface, pointing to a perforin- and secretion-dependent mechanism of muscle fiber injury.

Base Sequence↗
PubMed1996

Associations among PH and SH3 domain-containing proteins and Rho-type GTPases in Yeast.

The src homology region 3 (SH3) domain-bearing protein Bem1p and the Rho-type GTPase Cdc42p are important for bud emergence in Saccharomyces cervisiae. Here, we present evidence that through its second SH3 domain, Bem1p binds to the structurally and functionally similar proteins Boi1p and Boi2p, each of which contain an SH3 and pleckstrin homology (PH) domain. Deletion of BOI1 and BO12 together leads to impaired morphogenesis and poor ability. A PH domain-bearing segment of Boi1p that lacks the Bem1p-binding site is necessary and sufficient for function. This segment of Boi1p displays a two-hybrid interaction with Cdc42p, suggesting that Boi1p either binds directly to or is part of a larger complex that contains Cdc42p. Consistent with these possibilities, overexpression of Boi1p inhibits bud emergence, but this inhibition is counteracted by cooverexpression of Cdc42p. Increased expression of the Rho-type GTPase Rho3p, which is implicated in bud growth defects of boil boi2 mutants, suggesting that Boi1p and Boi2p may also play roles in the activation or function of Rho3p. These findings provide an example of a tight coupling in function between PH domain-bearing proteins and both Rho-type GTPases and SH3 domain-containing proteins, and they raise the possibility that Boi1p and Boi2 play a role in linking the actions of Cdc42p and Rho3p.

Adaptor Proteins, Signal Transducing↗
PubMed1996

Interactions between the ankyrin repeat-containing protein Akr1p and the pheromone response pathway in Saccharomyces cerevisiae.

Akr1p, which contains six ankyrin repeats, was identified during a screen for mutations that displayed synthetic lethality with a mutant allele of the bud emergence gene BEM1. Cells from which AKR1 had been deleted were alive but misshapen at 30 degrees C and inviable at 37 degrees C. During a screen for mutants that required one or more copies of wild-type AKR1 for survival at 30 degrees C, we isolated mutations in GPA1, which encodes the G alpha subunit of the pheromone receptor-coupled G protein. (The active subunit of this G protein is G beta gamma, and G alpha plays an inhibitory role in G beta gamma-mediated signal transduction.) AKR1 could serve as a multicopy suppressor of the lethality caused by either loss of GPA1 or overexpression of STE4, which encodes the G beta subunit of this G protein, suggesting that pheromone signaling is inhibited by overexpression of Akr1p. Mutations in AKR1 displayed synthetic lethality with a weak allele of GPA1 and led to increased expression of the pheromone-inducible gene FUS1, suggesting that Akr1p normally (and not just when overexpressed) inhibits signaling. In contrast, deletion of BEM1 resulted in decreased expression of FUS1, suggesting that Bem1p normally facilitates pheromone signaling. During a screen for proteins that displayed two-hybrid interactions with Akr1p, we identified Ste4p, raising the possibility that an interaction between Akr1p and Ste4p contributes to proper regulation of the pheromone response pathway.

Amino Acid Sequence↗
PubMed1996

Identification of the bud emergence gene BEM4 and its interactions with rho-type GTPases in Saccharomyces cerevisiae.

The Rho-type GTPase Cdc42p is required for cell polarization and bud emergence in Saccharomyces cerevisiae. To identify genes whose functions are linked to CDC42, we screened for (i) multicopy suppressors of a Ts- cdc42 mutant, (ii) mutants that require multiple copies of CDC42 for survival, and (iii) mutations that display synthetic lethality with a partial-loss-of-function allele of CDC24, which encodes a guanine nucleotide exchange factor for Cdc42p. In all three screens, we identified a new gene, BEM4. Cells from which BEM4 was deleted were inviable at 37 degrees C. These cells became unbudded, large, and round, consistent with a model in which Bem4p acts together with Cdc42p in polarity establishment and bud emergence. In some strains, the ability of CDC42 to serve as a multicopy suppressor of the Ts- growth defect of deltabem4 cells required co-overexpression of Rho1p, which is an essential Rho-type GTPase necessary for cell wall integrity. This finding suggests that Bem4p also affects Rho1p function. Bem4p displayed two-hybrid interactions with Cdc42p, Rho1p, and two of the three other known yeast Rho-type GTPases, suggesting that Bem4p can interact with multiple Rho-type GTPases. Models for the role of Bem4p include that it serves as a chaperone or modulates the interaction of these GTPases with one or more of their targets or regulators.

Amino Acid Sequence↗
PubMed1995

Inactivated influenza virus, when presented on dendritic cells, elicits human CD8+ cytolytic T cell responses.

Inactivated or subunit virus preparations have been excellent vaccines for inducing antibody responses. Generation of cytolytic T cell responses, however, is thought to require replicating virus, primarily to provide sufficiently large amounts of cytoplasmic proteins for processing and presentation on major histocompatibility complex class I molecules by antigen-presenting cells. Potent human CD8+ cytolytic T cell responses to live replicating influenza A virus are generated when dendritic cells are used as the antigen-presenting cells. Here, we demonstrate that dendritic cells pulsed with poorly replicating, heat- or ultraviolet-inactivated influenza virus, induce equally strong CD8+ cytolytic T lymphocyte responses. The cytotoxic T lymphocytes are generated in the apparent absence of CD4+ helper cells or exogenous cytokines. Active viral protein synthesis is not required to charge class I molecules on dendritic cells. When pulsed with inactivated virus, < 1% of dendritic cells express nonstructural protein 1, which is only synthesized in the infectious cycle. To be optimally effective, however, the inactivated virus must retain its fusogenic activity, and presumably access the cytoplasm of dendritic cells. The data indicate, therefore, that dendritic cells require only small amounts of viral protein to charge class I molecules, most likely via traditional class I processing pathways. These results reopen the potential use of inactivated virus preparations as immunogens for cytotoxic T lymphocyte responses.

Antigen-Presenting Cells↗
PubMed1995

T cell receptor repertoire in polymyositis: clonal expansion of autoaggressive CD8+ T cells.

In polymyositis (PM), CD8+ T cell receptor (TCR) alpha/beta + cells invade and destroy major histocompatibility complex class I-positive muscle fibers. We combined polymerase chain reaction (PCR) and double-fluorescence immunocytochemistry to analyze the T cell receptor (TCR) repertoire expressed in muscle of PM patients. In patient 1, inverse PCR revealed a preferential usage of TCR V alpha 33.1, V beta 13.1, and V beta 5.1. Six of six TCR V alpha 33.1+ clones and five of seven V beta 13.1+ clones had identical nucleotide sequences. In contrast, the V beta 5.1+ TCRs were more heterogeneous. Similar results were obtained with an independent PCR method using primers specific for TCR V alpha 33, V beta 13, or V beta 5. No TCR sequences could be amplified from noninflammatory control muscle. Furthermore, none of the TCR sequences found in PM muscle could be detected in blood from the same patient or from a normal control subject. Immunohistochemistry confirmed that V beta 5.1 and V beta 13.1 were overrepresented in the muscle lesions of this patient. 32% of all CD8+ T cells were V beta 13.1+, and 16% were V beta 5.1+. However, approximately 60% of the CD8+ T cells that invaded muscle fibers were V beta 13.1+, whereas 10% were V beta 5.1+. In patient 2, 50% of the T cells were V beta 5.1+, and as in patient 1, these T cells were mainly located in interstitial areas. In patient 3, > 75% of the autoinvasive T cells stained with an anti-V beta 3 mAb. Sequence analysis of 15 PCR clones amplified with a V beta 3-specific primer showed that 9 (60%) sequences were identical. The results suggest that (a) a strikingly limited TCR repertoire is expressed in PM muscle; (b) there is a dissociation between the TCR usage of autoinvasive and interstitial T cells; and (c) the autoinvasive T cells are clonally expanded.

Amino Acid Sequence↗
PubMed1995

Interactions among proteins involved in bud-site selection and bud-site assembly in Saccharomyces cerevisiae.

Bud formation in yeast involves the actions of the Ras-type GTPase Rsr1, which is required for the proper selection of the bud site, and the Rho-type GTPase Cdc42, which is necessary for the assembly of cytoskeletal structures at that site. The Cdc24 protein is required both for proper bud-site selection and bud-site assembly and has been recently shown to display guanine-nucleotide-exchange factor (GEF) activity toward Cdc42. Here, we demonstrate, using recombinant proteins, that Cdc24 can also bind directly to Rsr1. This binding has no effect on the ability of Rsr1 to undergo intrinsic or GEF-stimulated GDP-GTP exchange. However, Cdc24 can inhibit both the intrinsic and GTPase-activating protein-stimulated GTPase activity of Rsr1 and thereby acts as a GTPase-inhibitor protein for Rsr1. Cdc24 thus appears to bind preferentially to the activated form of Rsr1. The SH3 domain-containing bud-site assembly protein Bem1 also binds directly to Cdc24, and we show here that this interaction is inhibited by Ca2+. Neither Bem1 nor Cdc42 affects the GTPase-inhibitor protein activity of Cdc24 toward Rsr1, and neither Bem1 nor Rsr1 affects the GEF activity of Cdc24 toward Cdc42. Taken together, these results suggest that Cdc24 enables the direct convergence of a Ras-like protein (Rsr1) and a Rho-like protein (Cdc42) with the SH3-domain-containing protein (Bem1) and that independent domains of Cdc24 are responsible for these different interactions. These results also suggest that rather than directly controlling the GEF activity of Cdc24, the primary roles of Rsr1 and Bem1 might be to control the positioning of Cdc24 within the cell.

Cell Division↗
PubMed1995

Soluble interleukin-4 receptor in atopic children.

The levels of natural soluble interleukin-4 receptor (shuIL-4R) were determined in the peripheral blood of 29 children with stable asthma, 10 children with asthma and acute respiratory infection, 11 healthy children with acute airway infection and 31 healthy controls. Healthy controls revealed the highest levels (median 1,082 pg/ml, range 524-1,900 pg/ml); which differed significantly from levels obtained with the blood of stable asthmatics (p < 0.01, median 658 pg/ml, range 329-1288 pg/ml), patients with asthma and acute respiratory infection (p < 0.01, median 663 pg/ml, range 0-1,250 pg/ml) and patients with respiratory infection alone (p < 0.01, median 674 pg/ml, range 466-1,110 pg/ml). In contrast, there was no significant difference in shuIL-4R content of cord blood obtained from newborns with a high or low risk of atopy. Additional analysis of interleukin-4 receptor (huIL-4R) on cultured lymphocytes from 13 stable asthmatic children and 14 healthy children indicated higher expression on CD4 cells (p < 0.05, median 2.2%, range 0.8-7.8%) compared to healthy controls (median 1.3%, range 0.7-3.3%). Therefore, diminished shuIL-4R concentrations in plasma may be related to inflammatory states but not specifically to atopy. The results support the notion that huIL-4R expressed on the cell surface may be regulated differently from the soluble form.

Acute Disease↗
PubMed1995

Treatment of myasthenia gravis by immunoadsorption of plasma.

We treated 16 patients with moderately severe to severe generalized myasthenia gravis (MG) by immunoadsorption (perfusion through a resin that adsorbs proteins) of 2,500 ml plasma on each of four alternate days. Fourteen patients who completed treatment all had significant improvement in strength (6 excellent, 6 good, and 2 fair), which began a mean of 42 hours after the first immunoadsorption, reached a maximum 4 days after the fourth immunoadsorption (mean, 250% of baseline strength), and returned to baseline over a mean of 2 months. Thirty-seven grams of plasma proteins were removed during each immunoadsorption, which required no replacement, compared with 175 grams during plasma exchange, which requires replacement with albumin. Serum or plasma concentration of all proteins fell, more so for most of the larger proteins than for the smaller ones: acetylcholine receptor antibody (AChR Ab) fell to a mean of 23% of original level, fibrinogen to 26%, C4 to 29%, IgM to 33%, IgG to 35%, CH50 to 41%, C3 to 42%, IgA to 54%, and albumin to 76%. All proteins, including AChR Ab, returned to their original levels within 1 to 3 weeks after the last immunoadsorption, while improvement in strength lasted a mean of 6 weeks longer. One seronegative patient had excellent improvement lasting more than a month. Activated complement C5a and white blood cell count rose during each immunoadsorption, while activated complement C3a fell, and each returned to its original level within hours. Eight patients had transient symptomatic hypotension attributable to withdrawal of blood more rapidly than it was returned; this hypotension was prevented or ameliorated by intravenous saline.(ABSTRACT TRUNCATED AT 250 WORDS)

Autoantibodies↗
PubMed1994

Control of the yeast bud-site assembly GTPase Cdc42. Catalysis of guanine nucleotide exchange by Cdc24 and stimulation of GTPase activity by Bem3.

Bud emergence in Saccharomyces cerevisiae involves cell cycle-regulated reorganizations of cortical cytoskeletal elements and requires the action of the Rho (Ras homologous)-type GTPase Cdc42. As a first step toward understanding how these cytoskeletal rearrangements are controlled, we have sought to identify those proteins that regulate the binding and hydrolysis of GTP by Cdc42. Here we report that the product of the CDC24 gene, which is required for proper bud-site selection and bud emergence, can stimulate the exchange of GTP for GDP on Cdc42. Combined with previously reported genetic data, this finding suggests that the processes of bud-site selection (which requires the action of a Ras-type GTPase) and bud-site assembly might be coordinated through an activator of a Rho-type GTPase. We also report the identification of a new gene, BEM3, that is a multicopy suppressor of the temperature-sensitive lethality caused by mutations in the bud emergence gene BEM2. Bem3 and Bem2 both contain a Rho GTPase-activating protein homology domain, but only Bem3 is able to stimulate the hydrolysis of GTP on Cdc42. These studies demonstrate that Cdc24 and Bem3 have GDP-release factor activity and GTPase-activating protein activity, respectively, toward the essential bud-site assembly GTPase Cdc42.

Amino Acid Sequence↗
PubMed1994

Characterization of a high molecular weight tumor necrosis factor-alpha mRNA in influenza A virus-infected macrophages.

Infection by influenza A virus has previously been shown to prime macrophages for a high TNF-alpha production. Influenza A virus induced a TNF-alpha mRNA accumulation that consisted of two types: a regular 1.7 kb and an additional high m.w. 2.4 kb species in murine macrophages, and a high m.w. 3.6 kb species in human monocytes. In this study, we further characterized this virus-induced, novel high m.w. TNF-alpha mRNA. The additional high m.w. TNF-alpha mRNA represented a true polyadenylated mRNA and its induction required exposure to infectious viruses. The regular and the high m.w. TNF-alpha mRNA were both found in the nuclear fraction and the cytoplasm. We excluded that the novel high m.w. TNF-alpha mRNA was an intron-containing precursor TNF-alpha mRNA that could have persisted in virus-infected macrophages. When TNF-alpha exons 1 to 4 and TNF-alpha exons 2 to 4 were amplified by polymerase chain reaction, only regular and no high m.w. bands were detected. By use of specific TNF-alpha intron I and intron III cDNA we could definitely demonstrate the absence of introns in the high m.w. TNF-alpha mRNA. The high m.w. TNF-alpha mRNA was free of TNF-beta and TNF intergenic region elements but contained the 5' and 3' untranslated region of TNF-alpha. Influenza A virus infection also induced a double band of IL-1 beta and IL-6 mRNA. Whether this novel high m.w. TNF-alpha mRNA represents a virus-induced abnormality or a superinduction of an otherwise normal but minor TNF-alpha transcript, and whether this high m.w. TNF-alpha mRNA species codes for a biologically active product, remains to be examined.

Animals↗
PubMed1994

Interactions between the bud emergence proteins Bem1p and Bem2p and Rho-type GTPases in yeast.

The SH3 domain-containing protein Bem1p is needed for normal bud emergence and mating projection formation, two processes that require asymmetric reorganizations of the cortical cytoskeleton in Saccharomyces cerevisiae. To identify proteins that functionally and/or physically interact with Bem1p, we screened for mutations that display synthetic lethality with a mutant allele of the BEM1 gene and for genes whose products display two-hybrid interactions with the Bem1 protein. CDC24, which is required for bud emergence and encodes a GEF (guanine-nucleotide exchange factor) for the essential Rho-type GTPase Cdc42p, was identified during both screens. The COOH-terminal 75 amino acids of Cdc24p, outside of the GEF domain, can interact with a portion of Bem1p that lacks both SH3 domains. Bacterially expressed Cdc24p and Bem1p bind to each other in vitro, indicating that no other yeast proteins are required for this interaction. The most frequently identified gene that arose from the bem1 synthetic-lethal screen was the bud-emergence gene BEM2 (Bender and Pringle. 1991. Mol. Cell Biol. 11:1295-1395), which is allelic with IPL2 (increase in ploidy; Chan and Botstein, 1993. Genetics. 135:677-691). Here we show that Bem2p contains a GAP (GTPase-activating protein) domain for Rho-type GTPases, and that this portion of Bem2p can stimulate in vitro the GTPase activity of Rho1p, a second essential yeast Rho-type GTPase. Cells deleted for BEM2 become large and multinucleate. These and other genetic, two-hybrid, biochemical, and phenotypic data suggest that multiple Rho-type GTPases control the reorganization of the cortical cytoskeleton in yeast and that the functions of these GTPases are tightly coupled. Also, these findings raise the possibility that Bem1p may regulate or be a target of action of one or more of these GTPases.

Adaptor Proteins, Signal Transducing↗
PubMed1994

Real-time identification of cerebral microemboli with US feature detection by a neural network.

PURPOSE: Abnormal transcranial Doppler ultrasonographic (US) signals indicating cerebral microembolism have characteristic but complex features. The authors wanted to assess the agreement among human observers and test the feasibility of an automated detection system. MATERIALS AND METHODS: Automated on-line detection of cerebral microemboli was accomplished by employing real-time overlapping Fourier transform and artificial neural network technology. By using long-term transcranial Doppler US recordings of the middle cerebral artery in consecutive cerebrovascular and cardiac patients, the method was evaluated in a clinical setting. RESULTS: The proportion of specific agreement (ps) among four experienced investigators identifying cerebral microemboli was high (mean ps, 0.91). Agreement among the neural network and the human observers was only slightly less (mean ps, 0.77). CONCLUSION: The technique allows highly reliable on-line evaluation of transcranial Doppler US recordings across multiple centers. It obviates time-consuming analyses by human observers.

Humans↗
PubMed1994

Influenza virus-infected dendritic cells stimulate strong proliferative and cytolytic responses from human CD8+ T cells.

Antigen-specific, CD8+, cytolytic T lymphocytes (CTLs) could potentially provide resistance to several infectious and malignant diseases. However, the cellular requirements for the generation of specific CTLs in human lymphocyte cultures are not well defined, and repetitive stimulation with antigen is often required. We find that strong CD8+ CTL responses to influenza virus can be generated from freshly isolated blood T cells, as long as dendritic cells are used as antigen presenting cells (APCs). Small numbers of dendritic cells (APC:T cell ratio of 1:50-1:100) induce these CTL responses from most donors in 7 d of culture, but monocytes are weak or inactive. Whereas both dendritic cells and monocytes are infected with influenza virus, the former serve as effective APCs for the induction of CD8+ T cells while the latter act as targets for the CTLs that are induced. The strong CD8+ response to influenza virus-infected dendritic cells is accompanied by extensive proliferation of the CD8+ T cells, but the response can develop in the apparent absence of CD4+ helpers or exogenous lymphokines. CD4+ influenza virus-specific CTLs can also be induced by dendritic cells, but the cultures initially must be depleted of CD8+ cells. These findings should make it possible to use dendritic cells to generate human, antigen-specific, CD8+ CTLs to other targets. The results illustrate the principle that efficient T cell-mediated responses develop in two stages: an afferent limb in which dendritic cells are specialized APCs and an efferent limb in which the primed T cells carry out an immune response to many types of presenting cells.

Antigen-Presenting Cells↗
PubMed1993

Biochemical comparisons of the Saccharomyces cerevisiae Bem2 and Bem3 proteins. Delineation of a limit Cdc42 GTPase-activating protein domain.

The Bem2 and Bem3 proteins, which appear to play roles in the regulation of bud site formation in Saccharomyces cerevisiae, show striking homology to a number of proteins that compose a family of GTPase-activating proteins (GAPs) for the rho-subgroup of ras-related GTP-binding proteins. These members include human platelet GAP for Cdc42Hs (the human homolog of a S. cerevisiae GTP-binding protein that regulates bud site assembly), the break point cluster region protein, the brain protein chimerin, the 85-kDa regulatory subunit (p85) of the phosphatidylinositol 3-kinase, and the ras-GAP-binding protein (p190). A fusion protein composed of the glutathione S-transferase protein and the rho-GAP homology region of Bem3 (designated GST-Bem3) stimulates the GTPase activity of the wild-type Cdc42Hs protein (Cdc42HsGly-12), but has no stimulatory effect on a GTPase-defective mutant (Cdc42HsVal-12), whereas a GST-Bem2 fusion protein does not stimulate the GTPase activity of either form of Cdc42Hs. We have compared the ability of GST-Bem3 to serve as a GAP for Cdc42Hs relative to other members of the rho-GAP subfamily and found the following order of potency: human platelet Cdc42Hs GAP > p190 > Bem3 > break point cluster region protein, whereas p85, like Bem2, shows no GAP activity or any ability to bind to the GTP-bound form of Cdc42Hs. We have taken advantage of the functional specificity exhibited by Bem3 (versus Bem2) in using Bem2/Bem3 chimeras, as well as different deletion mutant versions of the Bem3 protein, to delineate the limits of a functional Cdc42 GAP domain. The results of this study indicate that the carboxyl-terminal approximately 224 amino acids (which contain three regions of homology to the other members of the rho-GAP family) represent a "limit GAP." The first two appear to be important for binding to Cdc42Hs and for partial GAP activity.

Amino Acid Sequence↗
PubMed1993

Effect of granulocyte/macrophage colony-stimulating factor on human monocytes infected with influenza A virus. Enhancement of virus replication, cytokine release, and cytotoxicity.

The activating properties of granulocyte/macrophage (GM)-CSF were studied in vitro with human monocytes infected by influenza A virus. When monocytes were pretreated for 8 h with GM-CSF (100 U/ml) and then exposed to influenza A virus, de novo virus protein synthesis was enhanced, more virus particles were released, and cells were killed at a higher rate. In virus-infected monocytes, GM-CSF induced a more rapid IFN-alpha release and potentiated production of TNF-alpha, IL-1 beta, and IL-6. Although GM-CSF or influenza A virus were each capable of independently activating TNF-alpha, IL-1 beta, and IL-6 gene transcription, a combination of both induced a massive cytokine mRNA accumulation which was readily translated into bioactive protein. Thus, GM-CSF may display a Janus-like action by accelerating virus infection but also by priming monocytes for elevated cytokine production. Whether the facilitated influenza A virus replication caused by GM-CSF may be counterbalanced by an improved cytokine response remains to be studied under more complex in vivo conditions.

Base Sequence↗