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R Blumenthal

Publications and source records attributed to R Blumenthal.

At least 37 records · Page 2Linked to original sources
PubMed2000

P-glycoprotein-overexpressing multidrug-resistant cells are resistant to infection by enveloped viruses that enter via the plasma membrane.

The multidrug resistance gene product P-glycoprotein confers drug resistance to tumor cells by acting as a transporter that blocks the entry into the cell of a great variety of drugs and hydrophobic peptides. In this study we find that in drug-resistant cells, the insertion of the influenza virus fusion protein (hemagglutinin-2) into the plasma membrane is blocked and that the fusion of the viral envelope with the plasma membrane of these cells is impaired. Multidrug-resistant cells display significant resistance to infection by envelope viruses that invade cells by fusion with the plasma membrane, but not to infection by pH-dependent viruses that penetrate cells by fusion with endocytic vesicles. These observations suggest that multidrug resistance phenomena may protect cells from infection by a large group of disease-causing viruses that includes human immunodeficiency virus, herpes simplex virus, and some cancer-inducing retroviruses.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2000

Glycosphingolipids promote entry of a broad range of human immunodeficiency virus type 1 isolates into cell lines expressing CD4, CXCR4, and/or CCR5.

Treatment of human osteosarcoma cells, expressing CD4 and various chemokine receptors, with the glucosylceramide synthase inhibitor 1-phenyl-2-hexadecanoylamino-3-morpholino-1-propanol (PPMP), blocked target membrane glycosphingolipid (GSL) biosynthesis and reduced the susceptibility of cells to infection and fusion mediated by envelope glycoproteins from a variety of human immunodeficiency virus type 1 (HIV-1) isolates that utilize CXCR4 and/or CCR5. PPMP treatment of the cell lines did not significantly change the cell surface expression of CD4, CXCR4, and/or CCR5, nor did it alter the chemokine receptor association with CD4. PPMP-treated cells exhibited no changes in chemokine-induced Ca(2+) mobilization and chemotaxis. However, massive envelope glycoprotein conformational changes triggered by CD4 and the appropriate chemokine receptor on the target membrane were inhibited when the target cells were treated with PPMP. Addition of various purified GSLs to PPMP-treated target cells showed that for all isolates tested, globotriaosylceramide (Gb3) was the most potent GSL in restoring the fusion susceptibility of target cells with cells expressing HIV-1 envelope glycoproteins; addition of the monosialoganglioside GM3 yielded a slight enhancement of fusion susceptibility. Our data are consistent with the notion that a limited number of specific GSL species serve as crucial elements in organizing gp120-gp41, CD4, and an appropriate chemokine receptor into a membrane fusion complex.

3T3 Cells↗
PubMed1999

Human ECT2 is an exchange factor for Rho GTPases, phosphorylated in G2/M phases, and involved in cytokinesis.

Animal cells divide into two daughter cells by the formation of an actomyosin-based contractile ring through a process called cytokinesis. Although many of the structural elements of cytokinesis have been identified, little is known about the signaling pathways and molecular mechanisms underlying this process. Here we show that the human ECT2 is involved in the regulation of cytokinesis. ECT2 catalyzes guanine nucleotide exchange on the small GTPases, RhoA, Rac1, and Cdc42. ECT2 is phosphorylated during G2 and M phases, and phosphorylation is required for its exchange activity. Unlike other known guanine nucleotide exchange factors for Rho GTPases, ECT2 exhibits nuclear localization in interphase, spreads throughout the cytoplasm in prometaphase, and is condensed in the midbody during cytokinesis. Expression of an ECT2 derivative, containing the NH(2)-terminal domain required for the midbody localization but lacking the COOH-terminal catalytic domain, strongly inhibits cytokinesis. Moreover, microinjection of affinity-purified anti-ECT2 antibody into interphase cells also inhibits cytokinesis. These results suggest that ECT2 is an important link between the cell cycle machinery and Rho signaling pathways involved in the regulation of cell division.

Antibodies↗
PubMed1999

Role of glycosphingolipids in HIV-1 entry: requirement of globotriosylceramide (Gb3) in CD4/CXCR4-dependent fusion.

We have recently shown that addition of human erythrocyte glycosphingolipids (GSL) to non-human CD4+ or GSL-depleted human CD4+ cells rendered those cells susceptible to gp120-gp41-mediated cell fusion (Puri et al., BBRC, 1998). One GSL fraction (Fraction 3) isolated from human erythrocyte GSL mixture exhibited the highest recovery of fusion following incorporation into CD4+ non-human and GSL-depleted HeLa-CD4 cells (HeLa-CD4/GSL-). Structural analysis of Fraction 3 showed that this GSL had identical head group as the known GSL, Gal(alpha1-->4)Gal(beta1-->4)Glc-Ceramide (Gb3) (Puri et al., PNAS, 1998). Here we report that presence of Gb3 in CD4+/CXCR4+ cells but not CD4+/CXCR4 cells allows fusion with HIV-1Lai-envelope glycoprotein expressing cells (TF228). Therefore, Gb3 functions in conjunction with HIV-1 co-receptor, CXCR4 to promote fusion. We propose that Gb3 functions by recruiting CD4 and/or CXCR4 at the fusion site through structurally specific interactions.

CD4 Antigens↗
PubMed1999

Conformational intermediates and fusion activity of influenza virus hemagglutinin.

Three strains of influenza virus (H1, H2, and H3) exhibited similar characteristics in the ability of their hemagglutinin (HA) to induce membrane fusion, but the HAs differed in their susceptibility to inactivation. The extent of inactivation depended on the pH of preincubation and was lowest for A/Japan (H2 subtype), in agreement with previous studies (A. Puri, F. Booy, R. W. Doms, J. M. White, and R. Blumenthal, J. Virol. 64:3824-3832, 1990). While significant inactivation of X31 (H3 subtype) was observed at 37 degrees C at pH values corresponding to the maximum of fusion (about pH 5.0), no inactivation was seen at preincubation pH values 0.2 to 0.4 pH units higher. Surprisingly, low-pH preincubation under those conditions enhanced the fusion rates and extents of A/Japan as well as those of X31. For A/PR 8/34 (H1 subtype), neither a shift of the pH (to >5.0) nor a decrease of the temperature to 20 degrees C was sufficient to prevent inactivation. We provide evidence that the activated HA is a conformational intermediate distinct from the native structure and from the final structure associated with the conformational change of HA, which is implicated by the high-resolution structure of the soluble trimeric fragment TBHA2 (P. A. Bullough, F. M. Hughson, J. J. Skehel, and D. C. Wiley, Nature 371:37-43, 1994).

Anilino Naphthalenesulfonates↗
PubMed1999

Role of the membrane-proximal domain in the initial stages of human immunodeficiency virus type 1 envelope glycoprotein-mediated membrane fusion.

We have examined mutations in the ectodomain of the human immunodeficiency virus type 1 transmembrane glycoprotein gp41 within a region immediately adjacent to the membrane-spanning domain for their effect on the outcome of the fusion cascade. Using the recently developed three-color assay (I. Muñoz-Barroso, S. Durell, K. Sakaguchi, E. Appella, and R. Blumenthal, J. Cell Biol. 140:315-323, 1998), we have assessed the ability of the mutant gp41s to transfer lipid and small solutes from susceptible target cells to the gp120-gp41-expressing cells. The results were compared with the syncytium-inducing capabilities of these gp41 mutants. Two mutant proteins were incapable of mediating both dye transfer and syncytium formation. Two mutant proteins mediated dye transfer but were less effective at inducing syncytium formation than was wild-type gp41. The most interesting mutant proteins were those that were not capable of inducing syncytium formation but still mediated dye transfer, indicating that the fusion cascade was blocked beyond the stage of small fusion pore formation. Fusion mediated by the mutant gp41s was inhibited by the peptides DP178 and C34.

Amino Acid Sequence↗
PubMed1998

The neutral glycosphingolipid globotriaosylceramide promotes fusion mediated by a CD4-dependent CXCR4-utilizing HIV type 1 envelope glycoprotein.

Previously, we showed that the addition of human erythrocyte glycosphingolipids (GSLs) to nonhuman CD4(+) or GSL-depleted human CD4(+) cells rendered those cells susceptible to HIV-1 envelope glycoprotein-mediated cell fusion. Individual components in the GSL mixture were isolated by fractionation on a silica-gel column and incorporated into the membranes of CD4(+) cells. GSL-supplemented target cells were then examined for their ability to fuse with TF228 cells expressing HIV-1LAI envelope glycoprotein. We found that one GSL fraction, fraction 3, exhibited the highest recovery of fusion after incorporation into CD4(+) nonhuman and GSL-depleted HeLa-CD4 cells and that fraction 3 contained a single GSL fraction. Fraction 3 was characterized by MS, NMR spectroscopy, enzymatic analysis, and immunostaining with an antiglobotriaosylceramide (Gb3) antibody and was found to be Gal(alpha1-->4)Gal(beta1-->4)Glc-Cer (Gb3). The addition of fraction 3 or Gb3 to GSL-depleted HeLa-CD4 cells recovered fusion, but the addition of galactosylceramide, glucosylceramide, the monosialoganglioside, GM3, lactosylceramide, globoside, the disialoganglioside, GD3, or alpha-galactosidase A-digested fraction 3 had no effect. Our findings show that the neutral GSL, Gb3, is required for CD4/CXCR4-dependent HIV-1 fusion.

Acetylation↗
PubMed1998

A synthetic all D-amino acid peptide corresponding to the N-terminal sequence of HIV-1 gp41 recognizes the wild-type fusion peptide in the membrane and inhibits HIV-1 envelope glycoprotein-mediated cell fusion.

Recent studies demonstrated that a synthetic fusion peptide of HIV-1 self-associates in phospholipid membranes and inhibits HIV-1 envelope glycoprotein-mediated cell fusion, presumably by interacting with the N-terminal domain of gp41 and forming inactive heteroaggregates [Kliger, Y., Aharoni, A., Rapaport, D., Jones, P., Blumenthal, R. & Shai, Y. (1997) J. Biol. Chem. 272, 13496-13505]. Here, we show that a synthetic all D-amino acid peptide corresponding to the N-terminal sequence of HIV-1 gp41 (D-WT) of HIV-1 associates with its enantiomeric wild-type fusion (WT) peptide in the membrane and inhibits cell fusion mediated by the HIV-1 envelope glycoprotein. D-WT does not inhibit cell fusion mediated by the HIV-2 envelope glycoprotein. WT and D-WT are equally potent in inducing membrane fusion. D-WT peptide but not WT peptide is resistant to proteolytic digestion. Structural analysis showed that the CD spectra of D-WT in trifluoroethanol/water is a mirror image of that of WT, and attenuated total reflectance-fourier transform infrared spectroscopy revealed similar structures and orientation for the two enantiomers in the membrane. The results reveal that the chirality of the synthetic peptide corresponding to the HIV-1 gp41 N-terminal sequence does not play a role in liposome fusion and that the peptides' chirality is not necessarily required for peptide-peptide interaction within the membrane environment. Furthermore, studies along these lines may provide criteria to design protease-resistant therapeutic agents against HIV and other viruses.

Amino Acid Sequence↗
PubMed1998

Interferon-beta is required for interferon-alpha production in mouse fibroblasts.

The type I interferons--interferon-alpha (IFN-alpha) and interferon-beta (IFN-beta)--are critical for protection against viruses during the acute stage of viral infection [1,2]. Furthermore, type I interferons have been implicated as important mediators in the regulation of lymphocyte development [3], immune responses [4,5] and the maintenance of immunological memory of cytotoxic T cells [6,7]. The different IFN-alpha subtypes are encoded by 12 genes in the mouse [8] whereas IFN-beta is encoded by only one gene [9]. IFN-alpha and IFN-beta have a high degree of sequence homology and are thought to interact with the same surface receptor on target cells [10,11]. As an approach to analysing the different biological functions of IFN-alpha and IFN-beta, we have generated a mouse strain with an inactivated IFN-beta gene. We report here that embryonic fibroblasts from such mice produce neither IFN-beta nor IFN-alpha upon Sendal virus infection, whereas the production of IFN-alpha by leukocytes from the same strain of mice is intact. IFN-alpha production in embryonic fibroblasts from IFN-beta-/- mice could be rescued by 'priming' the cells using exogenous IFN-beta. These results imply a unique role for IFN-beta in the induction of type I interferons in peripheral tissues.

Animals↗
PubMed1998

Dilation of the human immunodeficiency virus-1 envelope glycoprotein fusion pore revealed by the inhibitory action of a synthetic peptide from gp41.

We have monitored fusion between cell pairs consisting of a single human immunodeficiency virus-1 (HIV-1) envelope glycoprotein-expressing cell and a CD4+ target cell, which had been labeled with both a fluorescent lipid in the membrane and a fluorescent solute in the cytosol. We developed a new three-color assay to keep track of the cell into which fluorescent lipids and/or solutes are redistributed. Lipid and solute redistribution occur as a result of opening a lipid-permissive fusion pore and a solute-permissive fusion pore (FPS), respectively. A synthetic peptide (DP178) corresponding to residues 643-678 of the HIV-1LAI gp120-gp41 sequence (Wild, C.T., D.C. Shugars, T.K. Greenwell, C.B. McDanal, and T.J. Matthews. 1994. Proc. Natl. Acad. Sci. USA. 91:12676-12680) completely inhibited FPS at 50 ng/ml, whereas at that concentration there was 20-30% fusion activity measured by the lipid redistribution. The differences detected in lipid mixing versus contents mixing are maintained up to 6 h of coculture of gp120-41-expressing cells with target cells, indicating that DP178 can "clamp" the fusion complex in the lipid mixing intermediate for very long time periods. A peptide from the NH2-terminal of gp41, DP107, inhibited HIV-1LAI gp120-gp41-mediated cell fusion at higher concentrations, but with no differences between lipid and aqueous dye redistribution at the different inhibitor concentrations. The inhibition of solute redistribution by DP178 was complete when the peptide was added to the fusion reaction mixture during the first 15 min of coculture. We have analyzed the inhibition data in terms of a fusion pore dilation model that incorporates the recently determined high resolution structure of the gp41 core.

Anti-HIV Agents↗
PubMed1998

Human erythrocyte glycolipids promote HIV-1 envelope glycoprotein-mediated fusion of CD4+ cells.

We examined the role of target membrane glycolipids in CD4-mediated HIV-1 fusion by altering the glycolipid levels in CD4+ cells. CD4+ human cells exhibited 50% reduction in extent of fusion with gp120-gp41 expressing cells (TF228) when grown in the presence of a glycolipid synthesis inhibitor PPMP. We added erythrocyte glycolipids (GL) to fusion-incompetent CD4+ non-human cells by influenza-hemagglutinin-mediated fusion between GL-containing liposomes and target cells. Human erythrocyte GL (HuGL)-modified CD4+ non-human cells became susceptible to fusion with TF228 cells. Transfer of bovine erythrocyte glycolipids (BoGL) to CD4+ non-human cells under similar conditions did not complement HIV-1 fusion. Furthermore, addition of HuGL, but not BoGL, to PPMP-inhibited cells rescued fusion to the original levels. Our observations demonstrate that human erythrocyte glycolipids promote CD4-mediated HIV-1 fusion and certain glycolipid(s) from human erythrocytes may serve as alternative and/or additional cofactors in HIV-1 entry.

Animals↗
PubMed1998

Conformational changes in cell surface HIV-1 envelope glycoproteins are triggered by cooperation between cell surface CD4 and co-receptors.

We have continuously measured CD4-induced conformational changes of cell surface-expressed human immunodeficiency virus type-1 envelope glycoprotein gp120-gp41 in situ using 4,4'-dianilino-1, 1'-binaphthyl-5,5'-disulfonic acid, a fluorescent probe that binds to hydrophobic groups. CD4-expressing human T cell lines induced significant and rapid conformational changes (<1 min delay) in gp120-gp41 from T cell-tropic strains, and little conformational changes in gp120-gp41 from macrophage-tropic strains, with equivalent levels of envelope expression. Conversely, CD4-expressing human macrophages induced significant and rapid conformational changes in gp120-gp41 from macrophage-tropic strains, and little conformational changes in gp120-gp41 from T cell-tropic strains. Thus, the conformational changes undergone by gp120-gp41, which lead to membrane fusion, are highly cooperative and require both receptor and co-receptor. We used a dye transfer assay to show that neither membrane lipid fusion or fusion pore formation can occur with host cells having different tropism from the envelope.

Anilino Naphthalenesulfonates↗
PubMed1998

Small molecule inhibitor of HIV-1 cell fusion blocks chemokine receptor-mediated function.

The intersection of the HIV and the chemokine fields began with the observation that HIV entry into cells could be blocked by certain chemokines. Subsequent work showed that HIV entry is dependent on the presence of specific chemokine receptors. These observations led us to evaluate a series of compounds, ureido analogs of distamycin previously reported to block HIV entry into cells in vitro, for chemokine antagonist activity. One of the distamycin analogs, 2,2'[4,4'-[[aminocarbonyl]amino]bis[N,4'-di[pyrrole-2-carboxamide- 1,1'-dimethyl]]-6,8 napthalenedisulfonic acid] hexasodium salt (NSC 651016), is shown here to inhibit syncytia formation and cell fusion. Mechanistic studies showed that this inhibition was not due to conformational changes in gp120-gp41 induced by target cell CD4 and chemokine co-receptor and was therefore not due to interference with binding of HIV-1. Additional mechanistic studies demonstrated that NSC 651016 inhibited chemokine binding to specific chemokine receptors, induced CXCR4 and CCR5 receptor internalization, and inhibited chemokine-induced chemotaxis by macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, RANTES, and stromal-derived factor-1alpha but not monocyte chemotactic protein-1. Thus, we describe a novel compound that inhibits in vivo replication of HIV-1 by down-regulation of co-receptors. These data lead us to propose that NSC 651016 may have in vivo anti-inflammatory activity.

Anti-HIV Agents↗
PubMed1998

Comparison of the Heart and Estrogen/Progestin Replacement Study (HERS) cohort with women with coronary disease from the National Health and Nutrition Examination Survey III (NHANES III)

BACKGROUND: The Heart and Estrogen/Progestin Replacement Study (HERS) is the first large clinical trial designed to test the efficacy of postmenopausal estrogen/progestin therapy for secondary prevention of coronary heart disease (CHD). To examine the representativeness of the HERS cohort to the general population of postmenopausal women with CHD, we compared the baseline cardiovascular risk factor data from HERS with similar data from women presumed to have CHD from the National Health and Nutrition Examination Survey (NHANES) III. METHODS: Age, race, and cardiovascular disease risk factors were compared in the 2763 postmenopausal women younger than 80 years old, with a uterus, and with documented CHD in HERS versus 145 similarly aged women with clinical or electrocardiographic evidence of CHD from phase I of NHANES III. RESULTS: There were fewer current smokers in HERS (13%) than in the NHANES cohort (21.7%, p = 0.05). Similarly, a history of hypertension was less prevalent in HERS (58.6%) than in the NHANES cohort (69.3%, p = 0.03). Women with fasting triglyceride levels >3.39 mmol/L or fasting glucose levels >16.6 mmol/L were excluded from HERS, resulting in fewer diabetics (22.9% vs 29.5%, p = 0.26) and lower serum triglyceride levels (1.88 mmol/L vs 2.25 mmol/L, p = 0.19) in HERS versus the NHANES cohort. Systolic and diastolic blood pressure, body mass index, physical activity, and total LDL and HDL cholesterol were not significantly different between the two groups. CONCLUSIONS: The HERS cohort had fewer CHD risk factors than women with myocardial infarction or angina in NHANES III, although comparison is hindered by differences in selection criteria. The many women with diabetes and hypertriglyceridemia in the NHANES cohort emphasizes the importance of testing strategies for secondary prevention of CHD in this high-risk subgroup.

Aged↗
PubMed1998

Perioperative renal dysfunction and cardiovascular anesthesia: concerns and controversies.

In patients with renal disease undergoing cardiovascular surgery, perioperative management continues to be a challenge. Traditional answers have turned into new questions with the introduction of new agents and the redesign of old techniques. For ARF prevention, early recognition of pending deleterious compensatory changes is critical. Theoretically, therapeutic intervention designed to prevent ischemic renal failure should be designed to preserve the balance between RBF and oxygen delivery on one hand and oxygen demand on the other. Maintenance of adequate cardiac output distribution to the kidney is determined by the relative ratio of renal artery vascular resistance to systemic vascular resistance. Indeed, it should not be surprising to learn that norepinephrine (despite its vasoconstricting effect) has been reported to have no deleterious renal effects in patients with low systemic vascular resistance. Until recently, strategies for the treatment of ARF have been directed to supportive care with dialysis (to allow tubular regeneration). Various therapeutic maneuvers have been introduced in an attempt to accelerate the recovery of glomerular filtration, including dialysis, nutritional regimens, and new pharmacologic agents. A recent small prospective trial of low-dose dopamine in the prophylaxis of ARF in patients undergoing abdominal aortic aneurysm repair showed no benefit in those patients receiving dopamine. Conversely, the effects of intravenous atrial natriuretic peptide in the treatment of patients with ARF appear to offer benefit in patients with oliguria. Among 121 patients with oliguric renal failure, 63% of those who received a 24-hour infusion of atrial natriuretic peptide required dialysis within 2 weeks compared with 87% who did not. Whether this effect will be borne out in the future remains to be determined. The administration of epidermal growth factor after induction of ischemic ARF in rats has been shown to enhance tubular regeneration and accelerate recovery of kidney function. Human growth factor administration has been shown to increase GFR 130% greater than baseline in patients with chronic renal failure, but no data for clinical ARF have been reported. In addition, there have been significant improvements in dialysis technology in the treatment of ARF. Modern dialysis uses bicarbonate as a buffer as opposed to acetate, which reduces cardiovascular instability, and has more precise regulation of volume removal. Dialysate profiles and temperatures improve hemodynamics and reduce intradialytic hypotension. Techniques of hemodialysis without anticoagulation have reduced bleeding complications. Finally, dialysis membranes activate neutrophils and complement less with the biocompatible membranes used today that reduce recovery time and dialysis treatment. Evidence indicates that activation of complement and neutrophils by older dialysis membranes caused a greater incidence of hypotension, adding to ischemic renal injury. It remains to be determined whether early and frequent dialysis with biocompatible membranes, as well as other therapeutic interventions, will increase the survival of patients with perioperative ARF.

Acute Kidney Injury↗