Biomedical subjects

J B Rubins

Publications and source records attributed to J B Rubins.

51 records · Page 3Linked to original sources

PubMed1995

Impact of Streptococcus pneumoniae bacteremia and human immunodeficiency virus type 1 on oral mucosal immunity.

To determine whether defects in mucosal immunity were associated with invasive disease caused by a mucosal pathogen, Streptococcus pneumoniae, levels of salivary immunoglobulins and nonspecific immune factors were compared in subjects with human immunodeficiency virus type 1 (HIV-1) infection and in HIV-1-seronegative subjects with and without pneumococcal bacteremia. The IgA2 subclass may be of particular importance because S. pneumoniae produces IgA1 protease, which cleaves IgA1 but not IgA2. Levels (37-56 micrograms/mL) and proportions (11%-17%) of IgA2 were similar among groups. Serotype-specific capsular salivary IgA was present in a minority of patients with acute bacteremia. Levels of lactoferrin were increased with bacteremia. Neither selective mucosal IgA2 deficiency nor impaired nonspecific upper respiratory mucosal responses were associated with invasive pneumococcal disease during HIV-1 infection; thus, other defects in mucosal cellular responses and systemic immunity may predispose HIV-1-infected patients to invasive pneumococcal disease.

AIDS-Related Opportunistic Infections↗

PubMed1995

Impaired natural immunity to pneumolysin during human immunodeficiency virus infection in the United States and Africa.

Human immunodeficiency virus (HIV) infection is associated with a significantly increased incidence of pneumococcal pneumonia and concomitant bacteremia. We hypothesized that the predisposition of HIV-infected patients to invasive pneumococcal infection may be related, in part, to an impaired immune response to the pneumococcal antigen pneumolysin (PLY) because PLY facilitates bacterial invasion. We measured serum anti-PLY antibodies in two separate populations of HIV-infected and HIV-seronegative controls, using both an enzyme-linked immunosorbent assay method and a functional assay of antibody inhibition of PLY-induced hemolysis and cytotoxicity. HIV-infected patients in the United States had significantly lower titers of anti-PLY antibodies by both methods than did seronegative control subjects. Moreover, HIV-infected patients in Kenya who later developed pneumococcal bacteremia also had significantly lower anti-PLY antibody levels at baseline compared with seronegative control subjects. We conclude that lower baseline levels of antibodies to PLY are associated with the higher incidence of bacteremic pneumococcal infections among HIV-infected patients.

AIDS-Related Opportunistic Infections↗

PubMed1995

Dual function of pneumolysin in the early pathogenesis of murine pneumococcal pneumonia.

Streptococcus pneumoniae is one of the most common etiologic agents of community-acquired pneumonia, particularly bacteremic pneumonia. Pneumolysin, a multifunctional cytotoxin, is a putative virulence factor for S. pneumoniae; however, a direct role for pneumolysin in the early pathogenesis of pneumococcal pneumonia has not been confirmed in vivo. We compared the growth of a pneumolysin-deficient (PLY[-]) type 2 S. pneumoniae strain with its isogenic wild-type strain (PLY[+]) after direct endotracheal instillation of bacteria into murine lungs. Compared with PLY(-) bacteria, infection with PLY(+) bacteria produced greater injury to the alveolar-capillary barrier, as assayed by albumin concentrations in alveolar lavage, and substantially greater numbers of PLY(+) bacteria were recovered in alveolar lavages and lung homogenates at 3 and 6 h after infection. The presence of pneumolysin also contributed to the development of bacteremia, which was detected at 3 h after intratracheal instillation of PLY(+) bacteria. The direct effects of pneumolysin on lung injury and on the ability of pneumococci to evade local lung defenses was confirmed by addition of purified recombinant pneumolysin to inocula of PLY(-) pneumococci, which promoted growth of PLY(-) bacteria in the lung to levels comparable to those seen with the PLY(+) strain. We further demonstrated the contributions of both the cytolytic and the complement-activating properties of pneumolysin on enhanced bacterial growth in murine lungs using genetically modified pneumolysin congeners and genetically complement-deficient mice. Thus, pneumolysin facilitates intraalveolar replication of pneumococci, penetration of bacteria from alveoli into the interstitium of the lung, and dissemination of pneumococci into the bloodstream during experimental pneumonia. Moreover, both the cytotoxic and the complement-activating activities of pneumolysin may contribute independently to the acute pulmonary injury and the high rates of bacteremia which characterize pneumococcal pneumonia.

Animals↗

PubMed1994

Anti-pneumolysin activity of commercially available alpha 1-antitrypsin is due to cholesterol impurities.

Pneumolysin (PLY), the principal cytolytic toxin of Streptococcus pneumoniae, may be important in the pathogenesis of acute lung injury during pneumococcal pneumonia. However, the local host defenses that limit PLY injury to lung tissues have not been characterized. We investigated the ability of a commercial preparation of alpha 1-antitrypsin (alpha 1-AT), a major plasma anti-proteinase, to inhibit PLY. At normal plasma concentrations, the alpha 1-AT preparation prevented PLY injury to bovine pulmonary artery endothelial cells, rat alveolar epithelial cells, and human erythrocytes. The alpha 1-AT preparation selectively inhibited thiol-activated bacterial toxins; it was inactive against snake venom hemolysins, mastoparan, and oxygen-stable bacterial toxins. Biochemical characterization of the alpha 1-AT preparation and comparison with other available alpha 1-AT preparations revealed that this inhibitory activity was due to contamination with nanomolar concentrations of cholesterol. Characterization of non-immune human plasma anti-pneumolysin activity showed that beta-lipoprotein fractions contain the major inhibitory activity. We caution other investigators that the inhibition of bacterial virulence by these alpha 1-AT preparations may indicate toxin-mediated, rather than protease-mediated, mechanisms.

Animals↗

PubMed1994

Pneumolysin activates phospholipase A in pulmonary artery endothelial cells.

Pneumolysin has been identified as a virulence factor in Streptococcus pneumoniae disease. In addition to producing tissue injury through its cytolytic effect, pneumolysin might injure tissues indirectly by eliciting an inflammatory response. We demonstrate for the first time that pneumolysin is a rapid and potent activator of cellular phospholipase A in bovine pulmonary artery endothelial cells. In contrast to other toxin-activated phospholipases, pneumolysin-stimulated phospholipase A showed no substrate specificity among major cellular membrane phospholipids. Phospholipase A activation required the formation of functional transmembrane pores by pneumolysin rather than membrane lipid perturbation. Pneumolysin stimulation of phospholipase A was calcium dependent; however, pneumolysin did not appear to function simply as a calcium ionophore. Pneumolysin was capable of stimulating purified bee and snake venom phospholipase A2s against a phospholipid substrate isolated from endothelial cells. Thus, pneumolysin stimulates cellular phospholipase A and the resulting products might further injure tissues by direct cytolytic effect or by evoking inflammatory responses.

Animals↗

PubMed1993

Identification of hydrogen peroxide as a Streptococcus pneumoniae toxin for rat alveolar epithelial cells.

Streptococcus pneumoniae infections of the lung are associated with significant damage to the alveolar epithelium. Host phagocytes and pneumolysin, a cytolytic toxin of S. pneumoniae, are believed to contribute to this cellular damage, yet experiments in which these elements are absent demonstrate the presence of an additional soluble S. pneumoniae factor that is toxic to alveolar epithelium. We examined the effects of S. pneumoniae-associated alveolar epithelial cell injury by factors other than S. pneumoniae-derived pneumolysin or phagocyte products by exposing cultured rat type II alveolar epithelial cells (RAEC) to S. pneumoniae mutants that lacked pneumolysin activity. We found that mutant pneumolysin-deficient strains of S. pneumoniae produced injury to RAEC similar to that produced by the parent strains. A toxin of type 14 S. pneumoniae was distinguished from pneumolysin by physiochemical (i.e., molecular mass and heat stability) and functional (i.e., hemolytic activity and cytotoxic activity) properties and was identified as hydrogen peroxide. All S. pneumoniae strains tested produced hydrogen peroxide, and in many strains hydrogen peroxide production was comparable to that of activated neutrophils. We conclude that S. pneumoniae produces hydrogen peroxide in concentrations that are cytotoxic to RAEC in vitro and that alveolar epithelial damage due to hydrogen peroxide may be involved in the pathogenesis of host cellular injury in pneumococcal pneumonia.

Animals↗

PubMed1993

Toxicity of pneumolysin to pulmonary alveolar epithelial cells.

Mortality during the first several days of pneumococcal pneumonia has not decreased appreciably over the past 30 years, despite the widespread use of antibiotics. Disruption of the alveolar epithelial barrier is likely an initial step in the pathogenesis of pneumococcal pneumonia. We report that soluble factors from Streptococcus pneumoniae can directly injure isolated rat alveolar epithelial cells. Using biochemical and immunological techniques, we identified pneumolysin as a major soluble S. pneumoniae toxin for alveolar epithelial cells. Alveolar epithelial cells at 24 or 72 h after isolation were equally sensitive to injury by purified pneumolysin. Purified pneumolysin substantially increased alveolar permeability in an isolated perfused rat lung model. Electron microscopy revealed that instilled pneumolysin caused widespread lung injury, primarily to type I alveolar epithelial cells. Pneumolysin toxicity to alveolar epithelial cells may be important in the pathogenesis of acute lung injury during pneumococcal pneumonia and may facilitate pneumococcal bacteremia.

Animals↗

PubMed1992

Toxicity of pneumolysin to pulmonary endothelial cells in vitro.

Pneumococcal pneumonia and bacteremia are associated with appreciable mortality, most of which occurs very early in the course of infection. An initial step in the pathogenesis of pneumococcal pneumonia may include disruption of the pulmonary endothelial barrier with subsequent alveolar hemorrhage. We sought to determine whether soluble factors from Streptococcus pneumoniae can directly injure pulmonary endothelial cells in vitro and to identify pneumococcal toxins that may be involved in endothelial cell injury. Suspensions of S. pneumoniae (10(8) organisms per ml) caused significant injury to cultured bovine pulmonary artery endothelial cells in a time-dependent manner. The degree of endothelial cell cytotoxicity differed among S. pneumoniae strains; among the strains tested, a type 14 strain was the most cytotoxic and a type 3 strain was the least cytotoxic. During autolysis, type 14 S. pneumoniae released a soluble endothelial cell cytotoxin that was distinct from S. pneumoniae capsular and cell wall polysaccharides. The soluble cytotoxin was further characterized as a thiol-activated, heat-sensitive protein that coeluted with purified pneumolysin during gel filtration. The identity of the S. pneumoniae endothelial cell cytotoxin as pneumolysin was further supported by the ability of purified pneumolysin and the inability of S. pneumoniae mutants which lack pneumolysin to injure endothelial cells, as well as by the inhibition of the soluble S. pneumoniae cytotoxin by a neutralizing antibody to pneumolysin. Pneumolysin appears to be the major S. pneumoniae soluble cytotoxin for pulmonary endothelial cells in vitro and may be an important factor in the pathogenesis of alveolar hemorrhages in S. pneumoniae infections.

Animals↗

PubMed1992

Identification of ras and ras-related low-molecular-mass GTP-binding proteins associated with rat lung lamellar bodies.

Recent evidence from genetic experiments in yeast and from studies using guanosine triphosphate (GTP) analogues in mammalian cells suggests a key role for low-molecular-mass GTP-binding proteins (LMM-GBPs) (Mr 19 to 28 kD) in processes of intracellular vesicular sorting and secretion. Assembly and exocytosis of the lamellar body (LB), the secretory organelle of the pulmonary alveolar type 2 pneumocyte, may be regulated by LMM-GBPs. We used [alpha-32P]GTP binding to Western blotted proteins, ultraviolet crosslinking of [alpha-32P]GTP to membrane proteins, immunoblotting with specific antisera, and botulinum exoenzyme C3-catalyzed ADP ribosylation to detect LMM-GBPs in LB. With the first two techniques, we have identified six LMM-GBPs of approximately 27, 25.5, 24.5, 23, 22, and 21 kD that are enriched in a highly purified LB fraction compared with type 2 pneumocyte homogenate, crude membranes, and cytosol. Further characterization of the LB LMM-GBPs by immunoblotting revealed that ras p21 is greatly enriched in the LB fraction compared with other type 2 pneumocyte fractions. In addition, botulinum exoenzyme C3 catalyzed the ADP ribosylation of 20- to 21-kD proteins that were similarly enriched in the LB fraction. In contrast, a monospecific antibody to ADP-ribosylation factor reacted with a 19-kD protein only in the type 2 pneumocyte homogenate and cytosol fractions. Monospecific antibodies to yeast Sec4 protein and to rab 3A did not react with any type 2 pneumocyte proteins. The LMM-GBPs specifically associated with LB may participate in intracellular events required for surfactant packaging and secretion.

Adenosine Diphosphate Ribose↗

PubMed1991

Mechanisms of mastoparan-stimulated surfactant secretion from isolated pulmonary alveolar type 2 cells.

Mastoparan, a tetradecapeptide component of wasp venom, is a potent activator of secretion in a variety of cell types, and has been shown to activate purified G-proteins reconstituted into phospholipid vesicles with a preferential activation of Gi over Gs (Higashijima, T., Uzu, S., Nakajima, T., and Ross, E. R. (1988) J. Biol. Chem. 263, 6491-6494). To identify the biochemical activities of mastoparan in a cellular system, we characterized the effects of mastoparan on signal transduction pathways in rat pulmonary alveolar type 2 epithelial cells, which synthesize and secrete pulmonary surfactant. Mastoparan inhibited adenylylcyclase activity in a manner that was dose-dependent (IC50 = 30 microM), but sensitive to neither guanine nucleotide nor pertussis toxin (PT). Mastoparan induced a PT-sensitive increase in cellular inositol trisphosphate and a rapid rise in cytosolic calcium released from intracellular stores; the time to onset of the calcium rise, but neither the rate nor the amplitude of the rise, were PT-sensitive. Mastoparan also caused a dose- (EC50 = 16 microM) and time-dependent activation of arachidonic acid release that was completely insensitive to pretreatment with PT. Secretion of pulmonary surfactant was increased by mastoparan approximately 8-fold over constitutive levels at 1 h with an EC50 = 20 microM, and mastoparan-stimulated secretion was partially sensitive to PT at late time points and to inhibitors of arachidonic acid metabolism, but not to the protein kinase C inhibitor H7. These findings are consistent with the activation of Gi proteins in type 2 cells by mastoparan, although the lack of predicted triphosphoguanine nucleotide and PT sensitivity for some activities indicates that mastoparan does not act in a manner strictly analogous to liganded receptors or that some activities are not mediated by activation of Gi. While mastoparan is a potent secretagogue in several cell types, its secretory activity appears to have only a limited dependence on the activation of Gi proteins in type 2 cells.

Animals↗

PubMed1991

Modification of brain guanine nucleotide-binding regulatory proteins by tryptamine-4,5-dione, a neurotoxic derivative of serotonin.

We have recently characterized a novel oxidation product of serotonin (5-hydroxytryptamine, 5-HT), tryptamine-4,5-dione, which increases 5-HT efflux from striatum and hippocampus and causes selective neuronal death. Exposure of striatal synaptosomes or the major brain guanine nucleotide-binding regulatory proteins Gi and Go to [3H]tryptamine-4,5-dione resulted in the radiolabeling of a major band with an apparent molecular mass equivalent to that of the alpha subunits of Gi and Go (approximately 40,000). The binding of [35S]guanosine-5'-O-(3-thiotriphosphate) ([35S]GTP-gamma-S) to Gi and Go and pertussis toxin-catalyzed [32P]ADP-ribosylation of the G protein alpha subunits were both inhibited in a dose-dependent manner by tryptamine-4,5-dione. Thus, neurotoxins such as tryptamine-4,5-dione may exert their effects through specific interactions with G proteins.

Adenosine Diphosphate Ribose↗

PubMed1991

Characterization of rabbit ileal receptors for Clostridium difficile toxin A. Evidence for a receptor-coupled G protein.

The purpose of this study was to characterize the surface receptor for toxin A, the enterotoxin from Clostridium difficile, on rabbit intestinal brush borders (BB) and on rat basophilic leukemia (RBL) cells. Purified toxin A was radiolabeled using a modified Bolton-Hunter method to sp act 2 microCi/micrograms, with retention of full biologic activity. 3H-Toxin A bound specifically to a single class of receptors on rabbit BB and on RBL cells with dissociation constants of 5.4 x 10(-8) and 3.5 x 10(-8) M, respectively. RBL cells were highly sensitive to toxin A (cell rounding) and had 180,000 specific binding sites per cell, whereas IMR-90 fibroblasts were far less sensitive to toxin A and lacked detectable specific binding sites. Exposure of BB to trypsin or chymotrypsin significantly reduced 3H-toxin A specific binding. Preincubation of BB with Bandeirea simplicifolia (BS-1) lectin also reduced specific binding, and CHAPS-solubilized receptors could be immobilized with WGA-agarose. The addition of 100 nM toxin A accelerated the association of 35S-GTP gamma S with rabbit ileal BB, and preincubation of BB with the GTP analogues GTP gamma S or Gpp(NH)p, significantly reduced 3H-toxin A specific binding. Our data indicate that the membrane receptor for toxin A is a galactose and N-acetyl-glucosamine-containing glycoprotein which appears to be coupled to a G protein.

Animals↗

PubMed1990

Compartmentalization of low molecular mass GTP-binding proteins among neutrophil secretory granules.

Neutrophils contain several distinct classes of secretory granules that may sequentially fuse with the phagosome after the ingestion of particulates, or that may be differentially exocytosed after cellular activation with soluble stimuli. The exocytosis of neutrophil secretory granules has been shown to be GTP-dependent at a step distal to activation of the transductional G proteins. Inasmuch as ras-related low molecular mass GTP-binding proteins have been shown to play regulatory roles in vesicle sorting in the secretory pathway in yeast, the differential mobilization of neutrophil granules might be regulated by distinct GTP-binding proteins. We therefore explored the distribution and identity of low molecular mass GTP-binding proteins in neutrophil secretory granules and other subcellular fractions. After lysis by nitrogen cavitation, four highly resolved fractions were harvested from discontinuous Percoll gradients: a microsomal fraction enriched for plasma membranes, specific granules, primary granules, and cytosol. At least seven bands of distinct Mr were detected by probing protein blots with [32P]GTP. Microsomes contained a prominent GTP-binding band at 26 kDa and weaker ones at 24 and 22.5 kDa; specific granules contained bands at 26, 24, 22, and 20 kDa; primary granules showed bands at 24 and 23 kDa; cytosol showed strong bands at 23.5 and 19 kDa and a weak band at 26 kDa. Antiserum against ADP-ribosylation factor reacted strongly with the 19-kDa band in cytosol but with none of the membrane fractions. None of these proteins was recognized by antibodies against ras or against Sec4p. Botulinum exoenzyme C3 labeled bands of molecular mass 20 and 21 kDa in cytosol and microsomes that have distinct mobilities from all the blotted [32P]GTP-binding proteins. The highly compartmentalized subcellular distribution of the blotted [32P]GTP-binding proteins in neutrophils is consistent with a regulatory role in the differential mobilization of granule compartments during cellular activation.

Adenosine Diphosphate Ribose↗

PubMed1990

Subcellular distribution and characterization of GTP-binding proteins in human neutrophils.

The subcellular distribution of GTP binding proteins in human neutrophils and their functional coupling to the N-formylmethionylleucylphenylalanine (FMLP) receptor was characterized to provide insight into mechanisms of cellular activation. Human neutrophils were nitrogen cavitated and fractionated on discontinuous Percoll gradients. Four subcellular fractions were obtained: cytosol, light membranes enriched for plasma membranes, specific granules and azurophilic granules. ADP-ribosylation catalyzed by pertussis toxin (PT) revealed a major substrate of 40 kDa only in plasma membrane and cytosol, and antiserum specific for Gi alpha confirmed the presence of neutrophil Gi alpha in plasma membrane and cytosol and its absence from specific granules. The cytosolic PT substrate was shown to be mostly in monomeric form by molecular sieve chromatography. The rate of the ribosyltransferase reaction was several-fold lower in cytosol compared to plasma membranes, and the extent of ADP-ribosylation was greatly augmented by supplementation with beta gamma subunits in cytosol. ADP-ribosylation catalyzed by cholera toxin (CT) revealed substrates of 52, 43 and 40 kDa in plasma membrane alone. FMLP receptors in plasma membrane were shown to be coupled to the 40 kDa substrate for CT by ligand-modulation of ADP-ribosylation, while FMLP added to specific granules did not induce ribosylation of this substrate even though FMLP receptors were found in high density in this compartment. Both 24 and 26 kDa [32P]GTP binding proteins were found to codistribute with FMLP receptors in specific granules and plasma membranes. Functional evidence for the coupling of GTP binding proteins to the FMLP receptor in specific granules was obtained by modulating [3H]FMLP binding with GTP gamma S, and by accelerating [35S]GTP gamma S binding with FMLP.

Cell Membrane↗

PubMed1990

GTP-binding proteins in rat liver nuclear envelopes.

Nuclear transport as well as reassembly of the nuclear envelope (NE) after completion of mitosis are processes that have been shown to require GTP and ATP. To study the presence and localization of GTP-binding proteins in the NE, we have combined complementary techniques of [alpha-32P]GTP binding to Western-blotted proteins and UV crosslinking of [alpha-32P]GTP with well-established procedures for NE subfractionation. GTP binding to blotted NE proteins revealed five low molecular mass GTP-binding proteins of 26, 25, 24.5, 24, and 23 kDa, and [alpha-32P]GTP photoaffinity labeling revealed major proteins with apparent molecular masses of 140, 53, 47, 33, and 31 kDa. All GTP-binding proteins appear to localize preferentially to the inner nuclear membrane, possibly to the interface between inner nuclear membrane and lamina. Despite the evolutionary conservation between the NE and the rough endoplasmic reticulum, the GTP-binding proteins identified differed between these two compartments. Most notably, the 68- and 30-kDa GTP-binding subunits of the signal recognition particle receptor, which photolabeled with [alpha-32P]GTP in the rough endoplasmic reticulum fraction, were totally excluded from the NE fraction. Conversely, a major 53-kDa photolabeled protein in the NE was absent from rough endoplasmic reticulum. Whereas Western-blotted NE proteins bound GTP specifically, all [alpha-32P]GTP photolabeled proteins could be blocked by competition with ATP, although with a competition profile that differed from that obtained with GTP. In comparative crosslinking studies with [alpha-32P]ATP, we have identified three specific ATP-binding proteins with molecular masses of 160, 78, and 74 kDa. The localization of GTP- and ATP-binding proteins within the NE appears appropriate for their involvement in nuclear transport and in the GTP-dependent fusion of nuclear membrane vesicles required for reassembly of the nucleus after mitosis.

Adenosine Triphosphate↗