Biomedical subjects

D G Russell

Publications and source records attributed to D G Russell.

At least 19 recordsLinked to original sources

PubMed1997

Golgi GDP-mannose uptake requires Leishmania LPG2. A member of a eukaryotic family of putative nucleotide-sugar transporters.

The synthesis of glycoconjugates within the secretory pathway of eukaryotes requires the provision of lumenal nucleotide-sugar substrates. This is particularly important for eukaryotic microbes such as Leishmania because they must synthesize considerable amounts of extracellular and cell surface glycoconjugates that play significant roles in the infectious cycle. Here we used properly oriented sealed microsomes to characterize lumenal uptake of GDP-Man in Leishmania donovani. In this system, GDP-Man uptake was saturable with an apparent Km for GDP-Man of 0.3 microM and facilitated its use as a donor substrate for lipophosphoglycan (LPG) synthesis. A lpg2(-) deletion mutant showed loss of GDP-Man but not UDP-Gal uptake, which was restored by introduction of the gene LPG2. Immunoelectron microscopy localized an active, epitope-tagged LPG2 protein to the Golgi apparatus. Thus, LPG2 is required for nucleotide-sugar transport activity and probably encodes this Golgi transporter. LPG2 belongs to a large family of eukaryotic genes that potentially encode transporters with different substrate specificities and/or cellular locations. In the future, the amenability of the Leishmania system to biochemical and genetic manipulation will assist in functional characterization of nucleotide-sugar transports from this and other eukaryotes. Furthermore, since LPG2 plays an important role in the Leishmania infectious cycle and mammalian cells lack a Golgi GDP-Man transporter, this activity may offer a new target for chemotherapy.

Amino Acid Sequence↗

PubMed1997

IL-6 produced by macrophages infected with Mycobacterium species suppresses T cell responses.

The ability of Mycobacterium bovis Calmette-Guérin bacillus-infected bone marrow-derived macrophages to process and present exogenously added Ags to T cells and stimulate their growth and production of IL-2 was examined. The infected macrophages were inhibited in their ability to activate T cells, and this inhibition could be transferred to uninfected macrophages with filtered supernatants from mycobacteria-infected macrophages. The inhibition was not due to decreases in macrophage viability, Ag uptake, or cell surface expression of MHC class II or other accessory molecules necessary for Ag presentation. Other intracellular pathogens such as Listeria monocytogenes and Leishmania mexicana did not induce the soluble inhibitory factor, while Mycobacterium avium strain 101 did, suggesting the factor is specific to infection with mycobacteria. The inhibitory effect was reversed completely by preincubation with neutralizing Abs against IL-6, and rIL-6 partially restored the effect. Approximately 10,000-fold more IL-6 was produced by mycobacteria-infected macrophages compared with uninfected controls. Such sustained levels of IL-6 may account for the immune unresponsiveness apparent in both human and murine mycobacterial disease.

Animals↗

PubMed1997

Transfer of phagocytosed particles to the parasitophorous vacuole of Leishmania mexicana is a transient phenomenon preceding the acquisition of annexin I by the phagosome.

The eukaryotic intracellular pathogen Leishmania mexicana resides inside macrophages contained within a membrane bound parasitophorous vacuole which, as it matures, acquires the characteristics of a late endosomal compartment. This study reports the selectivity of fusion of this compartment with other particle containing vacuoles. Phagosomes containing zymosan or live Listeria monocytogenes rapidly fused with L. mexicana parasitophorous vacuoles, while those containing latex beads or heat killed L. monocytogenes failed to do so. Fusigenicity of phagosomes was not primarily dependent on the receptor utilized for ingestion, as opsonization with defined ligands could not overcome the exclusion of either latex beads or heat killed organisms. However modulation of intracellular pH by pharmacological agents such as chloroquine and ammonium chloride increased delivery of live Listeria and also induced transfer of previously excluded particles. The absence of fusion correlated with the acquisition of annexin I, a putative lysosomal targeting, molecule, on the phagosome membrane. We propose that the acquisition of cellular membrane constituents such as annexin I during phagosome maturation can ultimately direct the fusion pathway of the vesicles formed and have described a model system to further document changes in vesicle fusigenicity within cells.

Animals↗

PubMed1996

Mycobacterium-containing phagosomes are accessible to early endosomes and reflect a transitional state in normal phagosome biogenesis.

The success of Mycobacterium as a pathogen hinges on its ability to modulate its intracellular environment. Mycobacterium avium reside in vacuoles with limited proteolytic activity, maintain cathepsin D in an immature form and remain accessible to internalized transferrin. Artificial acidification of isolated phagosomes facilitated processing of cathepsin D, demonstrating that pH alone limits proteolysis in these vacuoles. Moreover, analysis of IgG-bead phagosomes at early time points during their formation indicates that these phagosomes also acquire LAMP 1 and cathepsin D prior to the accumulation of proton-ATPases, and are transiently accessible to sorting endosomes. This suggests that the anomolous distribution of endosomal proteins in M. avium-containing vacuoles results from their arrested differentiation in an early transitional stage through which all phagosomes pass.

Animals↗

PubMed1996

On the molecular mechanism of chloroquine's antimalarial action.

Chloroquine is thought to exert its antimalarial effect by preventing the polymerization of toxic heme released during proteolysis of hemoglobin in the Plasmodium digestive vacuole. The mechanism of this blockade has not been established. We incubated cultured parasites with subinhibitory doses of [3H]chloroquine and [3H] quinidine. These [3H]quinoline compounds became associated with hemozoin as assessed by electron microscope autoradiography and subcellular fractionation. In vitro, binding of [3H]quinoline inhibitors to the hemozoin chain depended on the addition of heme substrate. These data counter previous conclusions regarding the lack of quinoline association with hemozoin, explain the exaggerated accumulation of quinolines in the plasmodium digestive vacuole, and suggest that a quinoline heme complex incorporates into the growing polymer to terminate chain extension, blocking further sequestration of toxic heme.

Animals↗

PubMed1996

Mycobacterium avium- and Mycobacterium tuberculosis-containing vacuoles are dynamic, fusion-competent vesicles that are accessible to glycosphingolipids from the host cell plasmalemma.

The vacuoles inhabited by viable Mycobacterium avium and Mycobacterium tuberculosis show limited fusion with endosomal and lysosomal compartments. This ability to regulate the maturation of their phagosomal compartments and restrict their differentiation into hydrolytically active vacuoles appears to correlate with the survival of the bacilli. Data presented in this current study demonstrate that despite the apparent isolation of mycobacterial vacuoles from the lysosomal network, they are dynamic, fusion-competent vesicles. Exploiting the ability of cholera toxin B subunit to bind to GM1 ganglioside on the macrophage plasmalemma, we demonstrate that these glycosphingolipids have ready access to the mycobacterial vacuoles. Entry into mycobacterial vacuoles is rapid, within 5 min of addition to the cells, and does not proceed through a brefeldin A-sensitive pathway. Furthermore, the gangliosides follow a route that differs from that taken by fluid-phase markers. TLC analysis gangliosides isolated from Mycobacterium-containing vacuoles, and IgG-bead phagosomes reveal similar profiles. These data indicate that rather than being fusion incompetent, mycobacterial vacuoles are actually highly dynamic, fusion-competent vesicles that behave like an extension of the recycling endosomal apparatus.

Animals↗

PubMed1996

Nucleus-encoded histone H1-like proteins are associated with kinetoplast DNA in the trypanosomatid Crithidia fasciculata.

Kinetoplast DNA (kDNA), the mitochondrial DNA of trypanosomatids, consists of thousands of minicircles and 20 to 30 maxicircles catenated into a single large network and exists in the cell as a highly organized compact disc structure. To investigate the role of kinetoplast-associated proteins in organizing and condensing kDNA networks into this disc structure, we have cloned three genes encoding kinetoplast-associated proteins. The KAP2, KAP3, and KAP4 genes encode proteins p18, p17, and p16, respectively. These proteins are small basic proteins rich in lysine and alanine residues and contain 9-amino-acid cleavable presequences. Proteins p17 and p18 are closely related to each other, with 48% identical residues and carboxyl tails containing almost exclusively lysine, alanine, and serine or threonine residues. These proteins have been expressed as Met-His6-tagged recombinant proteins and purified by metal chelate chromatography. Each of the recombinant proteins is capable of compacting kDNA networks in vitro and was shown to bind preferentially to a specific fragment of minicircle DNA. Expression of each of these proteins in an Escherichia coli mutant lacking the HU protein rescued a defect in chromosome condensation and segregation in the mutant cells and restored a near-normal morphological appearance. Proteins p16, p17, and p18 have been localized within the cell by immunofluorescence methods and appear to be present throughout the kDNA. Electron-microscopic immunolocalization of p16 shows that p16 is present both within the kDNA disc and in the mitochondrial matrix at opposite edges of the kDNA disc. Our results suggest that nucleus-encoded H1-like proteins may be involved in the organization and segregation of kDNA networks in trypanosomatids.

Animals↗

PubMed1996

Subcellular localization of Trypanosoma cruzi glycoprotein Gp72.

We have investigated the subcellular location of the Trypanosoma cruzi surface glycoprotein, Gp72, by introducing epitope-tagged copies of gp72 null-mutant cells. A tagged Gp72, containing three tandemly repeated copies of a human influenza hemagglutinin nonapeptide (HA) adjacent to the mature Gp72 amino terminus, was able to complement the null mutant phenotype, as well as being recognized in Western blots by both anti-HA antibody and the carbohydrate-specific monoclonal antibody WIC29.26. Integration of this epitope-tagged gp72 into the chromosomal gp72 locus produced a clonal cell line, 72HAN3.1G7, which was used for studies of the subcellular location of the epitope-tagged Gp72. Indirect immunofluorescence microscopy of fixed 72HAN3.1G7 epimastigotes showed that GP72 was evenly distributed over the cell body and somewhat concentrated in the proximal region of the flagellum. No fluorescence could be detected in the distal tip of the flagellum. Immunoelectron microscopy of fixed 72HAN3.1G7 epimastigotes revealed that Gp72 was predominantly membrane-associated and located on the cell surface. Indirect immunofluorescence microscopy of live 72HAN3.1G7 epimastigote cells showed a similar pattern of fluorescence on the flagellum, but no fluorescence was detected on the cell body, which was attributed to masking by other cell-surface components. Indirect immunofluorescence microscopy of fixed 72HAN3.1G7 amastigotes revealed that Gp72, which has long been considered to be expressed only in epimastigotes and metacyclic trypomastigotes, can be expressed in amastigotes, but it no longer contains the WIC29.26 carbohydrate epitope.

Amino Acid Sequence↗

PubMed1995

Of microbes and macrophages: entry, survival and persistence.

The macrophage is capable of fulfilling the roles of both host cell and effector cell in the immune clearance of many microbial infections. This dual capacity has led to the evolution of intriguing mechanisms whereby pathogens ensure the establishment and maintenance of intracellular infections. The strategies developed range from passive avoidance mechanisms to extremely active modulation of cell function. Recent advances have been made in our appreciation of the processes of intramacrophage parasitization from initial ligand-receptor interactions, through establishment of a stable intracellular environment, to the maintenance of a persistent infection.

Animals↗

PubMed1995

Differential targeting of two glucose transporters from Leishmania enriettii is mediated by an NH2-terminal domain.

Leishmania are parasitic protozoa with two major stages in their life cycle: flagellated promastigotes that live in the gut of the insect vector and nonflagellated amastigotes that live inside the lysosomes of the vertebrate host macrophages. The Pro-1 glucose transporter of L. enriettii exists as two isoforms, iso-1 and iso-2, which are both expressed primarily in the promastigote stage of the life cycle. These two isoforms constitute modular structures: they differ exclusively and extensively in their NH2-terminal hydrophilic domains, but the remainder of each isoform sequence is identical to that of the other. We have localized these glucose transporters within promastigotes by two approaches. In the first method, we have raised a polyclonal antibody against the COOH-terminal hydrophilic domain shared by both iso-1 and iso-2, and we have used this antibody to detect the transporters by confocal immunofluorescence microscopy and immunoelectron microscopy. The staining observed with this antibody occurs primarily on the plasma membrane and the membrane of the flagellar pocket, but there is also light staining on the flagellum. We have also localized each isoform separately by introducing an epitope tag into each protein sequence. These experiments demonstrate that iso-1, the minor isoform, resides primarily on the flagellar membrane, while iso-2, the major isoform, is located on the plasma membrane and the flagellar pocket. Hence, each isoform is differentially sorted, and the structural information for targeting each transporter isoform to its correct membrane address resides within the NH2-terminal hydrophilic domain.

Amino Acid Sequence↗

PubMed1995

Functional expression of a myo-inositol/H+ symporter from Leishmania donovani.

The vast majority of surface molecules in such kinetoplastid protozoa as members of the genus Leishmania contain inositol and are either glycosyl inositol phospholipids or glycoproteins that are tethered to the external surface of the plasma membrane by glycosylphosphatidylinositol anchors. We have shown that the biosynthetic precursor for these abundant glycolipids, myo-inositol, is translocated across the parasite plasma membrane by a specific transporter that is structurally related to mammalian facilitative glucose transporters. This myo-inositol transporter has been expressed and characterized in Xenopus laevis oocytes. Two-electrode voltage clamp experiments demonstrate that this protein is a sodium-independent electrogenic symporter that appears to utilize a proton gradient to concentrate myo-inositol within the cell. Immunolocalization experiments with a transporter-specific polyclonal antibody reveal the presence of this protein in the parasite plasma membrane.

Amino Acid Sequence↗

PubMed1995

Pressures in a simulated breast subjected to compression forces comparable to those of mammography. Work in Progress.

PURPOSE: To determine the magnitude and distribution of pressures in breast models subjected to mammographic compression forces. MATERIALS AND METHODS: A vinyl-wrapped, unleavened-bread-dough breast model was compressed. Pressures in the dough models were measured with a strain gauge connected to a water-filled sensor. RESULTS: Under broad compression, pressures were uniform (range, 2.76-22.06 kPa [0.4-3.2 psi]). Spot compression increased pressures by as much as 60%. Pressures dropped sharply outside of paddle margins. No pressure differentials were found near small, deep simulated masses. The pressure of large masses increased by 50% when subjected to full compression force. Vigorous digital palpation resulted in a pressure of more than 41.40 kPa (6 psi). CONCLUSION: General pressures were mild and uniform. Internal pressures varied inversely with the area to which force was applied. Pressure dissemination was restricted by the cohesive binder and allowed zones of greater pressure to form directly under the applied force. The surrounding matrix disseminated pressure and protected small masses; large masses, bearing the force of the paddle, were exposed to greater pressure. Digital palpation administered high pressures.

Biomechanical Phenomena↗

PubMed1994

Intracellular trafficking in Mycobacterium tuberculosis and Mycobacterium avium-infected macrophages.

Despite the potential role of the macrophage in the eradication of invading microbes, Mycobacterium species have evolved mechanisms to ensure their survival and replication inside the macrophage. Particles phagocytosed by macrophages normally will be delivered into acid lysosomal compartments for degradation. Mycobacterium must, in some way, avoid this fate by modulation of their phagosome. Immunoelectron microscopy of macrophages infected with Mycobacterium avium or Mycobacterium tuberculosis indicates that the vacuolar membrane surrounding the bacilli possesses the late endosomal/lysosomal marker, LAMP-1 (lysosomal-associated membrane protein-1), but lacks the vesicular proton-ATPase. Analysis of the intersection of the bacteria-containing vacuoles with the endocytic network of the macrophage supports previous studies indicating that these bacilli restrict the fusion capability of their intracellular compartments. The occurrence of vesicles containing lipoarabinomannan, discrete from those containing Mycobacterium, indicate that material does traffic out from the mycobacterial vacuole. To compensate for this loss of membrane, the vacuole must remain dynamic and fuse with LAMP-1-containing vesicles to maintain the density of this marker.

Animals↗

PubMed1994

A differentially expressed gene family encoding "amastin," a surface protein of Trypanosoma cruzi amastigotes.

A new family of closely related glycoproteins, collectively called amastins, has been found on the surface of the amastigote form of Trypanosoma cruzi. The gene family encoding these amastigote-specific proteins was identified by differentially screening an amastigote cDNA library with reverse transcribed poly(A)+ RNA from amastigote and epimastigote stages of the parasite. Amastins are encoded by eight or more tandem genes, at least five of which are distinguished by nucleotide point changes. The 1.4-kilobase amastin mRNAs are 50 times more abundant in amastigotes than in epimastigotes or trypomastigotes. The amastin genes are transcribed to an equal extent in both amastigotes and epimastigotes, indicating that the stage-specific amastin mRNA levels are determined by a post-transcriptional mechanism. Sequence determination of full-length cDNAs reveals an open reading frame encoding 174 amino acids and a 700-base pair 3'-untranslated region. Nascent amastins contain four distinct hydrophobic regions of 20-30 amino acids each, 2 at internal locations and 1 each at the N and C termini.

Amino Acid Sequence↗

PubMed1994

Identification of membrane-associated proteins in Trypanosoma brucei encoding an internal, EARLRAEE amino acid repeat.

Differential screening of expression libraries with nonimmune and day 4-6 immune serum from naive rats infected with the protozoan Trypanosoma lewisi was used to identify potential cell surface protein coding genes. Several T. lewisi cDNAs that resulted were partially characterized and used to clone the homologues from Trypanosoma brucei. The nucleotide sequence of the cDNAs encoding the Tb-29 genes of the parasitic protozoan T. brucei obtained by this method (3557 nucleotides for Tb-291 and 8729 nucleotides for Tb-292) encoded predicted open reading frames of 1070 and 2550 amino acids, respectively. The Tb-29 proteins encoded a large domain with an octapeptide (EARLRAEE) repeat (79 repeats in Tb-291 and 60 in Tb-292), which shared significant similarity with the octapeptide repeat of the S-antigen of isolate NF7 of Plasmodium falciparum. The predicted amino acid sequence of the Tb-292 protein encoded potential transmembrane domains (eight in total). Indirect immunofluorescence using confocal image analysis and immunoelectron microscopy located the EARLRAEE proteins to a membranous network. The Tb-29 proteins were most abundantly distributed to the area surrounding the nucleus, the region between the nucleus and the flagellar pocket, and the region immediately underneath the flagellar pocket membrane. The subcellular distribution of Tb-29 proteins suggests that these proteins may provide a constituent associated with the cell's vesicular transport system.

Amino Acid Sequence↗

PubMed1994

Lack of acidification in Mycobacterium phagosomes produced by exclusion of the vesicular proton-ATPase.

The success of Mycobacterium species as pathogens depends on their ability to maintain an infection inside the phagocytic vacuole of the macrophage. Although the bacteria are reported to modulate maturation of their intracellular vacuoles, the nature of such modifications is unknown. In this study, vacuoles formed around Mycobacterium avium failed to acidify below pH 6.3 to 6.5. Immunoelectron microscopy of infected macrophages and immunoblotting of isolated phagosomes showed that Mycobacterium vacuoles acquire the lysosomal membrane protein LAMP-1, but not the vesicular proton-adenosine triphosphatase (ATPase) responsible for phagosomal acidification. This suggests either a selective inhibition of fusion with proton-ATPase-containing vesicles or a rapid removal of the complex from Mycobacterium phagosomes.

Animals↗

PubMed1994

Molecular characterization and inhibition of a Plasmodium falciparum aspartic hemoglobinase.

Intraerythrocytic malaria parasites rapidly degrade virtually all of the host cell hemoglobin. We have cloned the gene for an aspartic hemoglobinase that initiates the hemoglobin degradation pathway in Plasmodium falciparum. It encodes a protein with 35% homology to human renin and cathepsin D, but has an unusually long pro-piece that includes a putative membrane spanning anchor. Immunolocalization studies place the enzyme in the digestive vacuole and throughout the hemoglobin ingestion pathway, suggesting an unusual protein targeting route. A peptidomimetic inhibitor selectively blocks the aspartic hemoglobinase, prevents hemoglobin degradation and kills the organism. We conclude that Plasmodium hemoglobin catabolism is a prime target for antimalarial chemotherapy and have identified a lead compound towards this goal.

Amino Acid Sequence↗

PubMed1994

Complement receptor-mediated uptake and tumor necrosis factor-alpha-mediated growth inhibition of Mycobacterium tuberculosis by human alveolar macrophages.

The relative phagocytosis and intracellular fate of Mycobacterium tuberculosis (MTB) (H37Ra) in human alveolar macrophages (AM) and their precursors blood monocytes (MN) was investigated. Uptake of MTB by MN and AM was confirmed by electron microscopy. At an infection ratio of 100:1 (MTB:target cell), the percentage of infected AM and the number of MTB per AM was > MN (p < 0.001, p < 0.0001, respectively). Uptake of MTB was increased by increasing concentrations of serum and decreased in the presence of heat-inactivated serum. Among complement receptors (CR) CR1, CR3, and CR4, the major CR mediating uptake of MTB by MN were CR1 and CR3, whereas for AM, CR4 was the major CR. When MN and AM were infected with MTB and cultured for up to 7 days, AM limited intracellular growth of MTB more effectively than MN as determined by a CFU assay. MTB stimulated production of TNF-alpha by mononuclear phagocytes and by AM > MN (p < 0.007). Pentoxifylline inhibited TNF-alpha production by mononuclear phagocytes and concurrently increased MTB growth (AM > MN). A polyclonal neutralizing antibody to TNF-alpha also increased MTB growth in AM. Thus, AM are more efficient in phagocytosis of MTB than MN, and uptake is mediated through CR4 to a greater extent than CR1 or CR3. The slowed replication of MTB in AM is associated with an increase in TNF-alpha production, and intracellular growth is promoted by pentoxifylline and neutralizing antibody to TNF-alpha. These data suggest that AM may play a prominent and efficient role in the primary defense of the lung in tuberculosis through CR-mediated uptake, predominantly CR4, and TNF-alpha-mediated killing of MTB.

Cells, Cultured↗