Effects of cytokines from activated immune cells on vascular cell growth and HIV-1 gene expression. Implications for AIDS-Kaposi's sarcoma pathogenesis.

Kaposi's sarcoma (KS) arises more frequently in homosexual and bisexual men than in other groups of HIV-1 infected individuals. Clinico-epidemiologic data indicate that homosexuals often are infected with multiple microbial agents and/or subjected to other antigenic stimuli, preceding or accompanying HIV-1 infection. Signs of immune activation, in fact, frequently have been detected in these individuals, and the onset of KS can precede any sign of immunodeficiency. These data have suggested that products from activated immune cells may affect the development of AIDS-KS. Here we report that conditioned media from activated or dysregulated T cells contain a variety of cytokines that promote the growth of spindle cells derived from KS lesions of AIDS patients (AIDS-KS cells) and induce normal vascular cells, potential cell progenitors of the AIDS-KS cells, to acquire features of the KS cell phenotype ("spindle" cell morphology and growth responsiveness to the mitogenic effect of extracellular HIV-1 Tat protein). The same conditioned media or cytokines promote HIV-1 gene expression and rescue defective HIV-1 proviruses, interrupting HIV-1 latency and increasing Tat production. The cellular and viral effects of cytokines are increased in an additive or synergistic manner by picomolar concentrations of extracellular Tat. These data suggest that cytokines produced by activated immune cells cooperate with HIV-1 infection in AIDS-KS pathogenesis.     

Diverse Bacteria Inhabit Living Hyphae of Phylogenetically Diverse Fungal Endophytes

Both the establishment and outcomes of plant-fungus symbioses can be influenced by abiotic factors, the interplay of fungal and plant genotypes, and additional microbes associated with fungal mycelia. Recently bacterial endosymbionts were documented in soilborne Glomeromycota and Mucoromycotina and in at least one species each of mycorrhizal Basidiomycota and Ascomycota. Here we show for the first time that phylogenetically diverse endohyphal bacteria occur in living hyphae of diverse foliar endophytes, including representatives of four classes of Ascomycota. We examined 414 isolates of endophytic fungi, isolated from photosynthetic tissues of six species of cupressaceous trees in five biogeographic provinces, for endohyphal bacteria using microscopy and molecular techniques. Viable bacteria were observed within living hyphae of endophytic Pezizomycetes, Dothideomycetes, Eurotiomycetes, and Sordariomycetes from all tree species and biotic regions surveyed. A focus on 29 fungus/bacterium associations revealed that bacterial and fungal phylogenies were incongruent with each other and with taxonomic relationships of host plants. Overall, eight families and 15 distinct genotypes of endohyphal bacteria were recovered; most were members of the Proteobacteria, but a small number of Bacillaceae also were found, including one that appears to occur as an endophyte of plants. Frequent loss of bacteria following subculturing suggests a facultative association. Our study recovered distinct lineages of endohyphal bacteria relative to previous studies, is the first to document their occurrence in foliar endophytes representing four of the most species-rich classes of fungi, and highlights for the first time their diversity and phylogenetic relationships with regard both to the endophytes they inhabit and the plants in which these endophyte-bacterium symbiota occur.Traits related to the establishment and outcome of plant-fungus symbioses can reflect not only abiotic conditions and the unique interactions of particular fungal and plant genotypes (49, 50, 56, 59, 62, 67) but also additional microbes that interact intimately with fungal mycelia (4, 12, 42). For example, mycorrhizosphere-associated actinomycetes release volatile compounds that influence spore germination in the arbuscular mycorrhizal (AM) fungus Gigaspora margarita (Glomeromycota) (14). Levy et al. (34) describe Burkholderia spp. that colonize spores and hyphae of the AM fungus Gigaspora decipiens and are associated with decreased spore germination. Diverse “helper” bacteria have been implicated in promoting hyphal growth and the establishment of ectomycorrhizal symbioses (23, 26, 57, 70). Minerdi et al. (43) found that a consortium of ectosymbiotic bacteria limited the ability of the pathogen Fusarium oxysporum to infect and cause vascular wilts in lettuce, with virulence restored to the pathogen when ectosymbionts were removed.In addition to interacting with environmental and ectosymbiotic bacteria, some plant-associated fungi harbor bacteria within their hyphae (first noted as “bacteria-like organisms” of unknown function) (38). These bacteria, best known from living hyphae of several species of the Glomeromycota and Mucoromycotina, can alter fungal interactions with host plants in diverse ways (see references 12, 31, and 51). For example, the vertically transmitted bacterium “Candidatus Glomeribacter gigasporarum” colonizes spores and hyphae of the AM fungus Gigaspora gigasporarum (9, 10). Removal of the bacterial partner from the fungal spores suppresses fungal growth and development, altering the morphology of the fungal cell wall, vacuoles, and lipid bodies (37). In turn, the discovery of phosphate-solubilizing bacteria within Glomus mossae spores (44), coupled with the recovery of a P-transporter operon in Burkholderia sp. from Gigaspora margarita (54), suggests a competitive role in phosphate acquisition and transport by these bacteria within the AM symbiosis. Within the Mucoromycotina, Partida-Martinez and Hertweck (51) reported that a soilborne plant pathogen, Rhizopus microsporus, harbors endosymbiotic Burkholderia that produces a phytotoxin (rhizoxin) responsible for the pathogenicity of the fungus.These examples, coupled with the discovery of bacteria within hyphae of the ectomycorrhizal Dikarya (Tuber borchii; Ascomycota; Laccaria bicolor and Piriformospora indica; Basidiomycota) (5-8, 58), suggest that the capacity to harbor endohyphal bacteria is widespread among fungi. To date, however, endocellular bacteria have been recovered only from fungi that occur in the soil and rhizosphere (12, 31). Here we report for the first time that phylogenetically diverse bacteria occur within living hyphae of foliar endophytic fungi, including members of four classes of filamentous Ascomycota. We use a combination of light and fluorescence microscopy to visualize bacterial infections within living hyphae of representative strains. Then, drawing from surveys of endophytes from asymptomatic foliage of cupressaceous trees in five biogeographic provinces, we provide a first characterization of the phylogenetic relationships, host associations, and geographic distributions of endohyphal bacteria associated with focal fungal endophytes.     

Metabolic pathways and nitrogen metabolism inLegionella pneumophila

An examination of cellular extracts ofLegionella pneumophila (Philadelphia 1 and Knoxville 1) was undertaken and key enzymes of the Embden-Meyerhof-Parnas (EMP) and pentose phosphate (PP) pathways, and the Krebs cycle were found. No enzymatic evidence of the ED pathway was obtained. In regard to carbon flow in the EMP pathway, the activities of fructose-1,6-biphosphatase (6–7.3 nmol/min/mg protein) and of phosphofructokinase (0.67–0.8 nmol/min/mg protein) suggested a gluconeogenic role. In further support of this direction, good activities were detected for phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxykinase. While an energized membrane was required for glutamate uptake by whole cells, an energized mechanism for glucose uptake could not be demonstrated. The Krebs cycle was essentially complete and, despite high specific activities for isocitrate and malate dehydrogenases, whole cells failed to oxidize these substrates, suggesting a transport deficiency. The major carbon and energy sources serine and glutamate were catabolized vial-serine dehydratase and glutamate-aspartate transaminase, respectively. This study confirmed that amino acids are catabolized via the Krebs cycle and that sugars are synthesized by the gluconeogenic enzymes of the EMP pathway.     

Characterization of the [4Fe-4S]+ cluster at the active site of aconitase by 57Fe, 33S, and 14N electron nuclear double resonance spectroscopy

57Fe, 33S, and 14N electron nuclear double resonance (ENDOR) studies have been performed to characterize the [4Fe-4S]+ cluster at the active site of aconitase. Q-band 57Fe ENDOLR of isotopically enriched enzyme, both substrate free and in the enzyme-substrate complex, reveals four inequivalent iron sites. In agreement with M?ssbauer studies [Kent et al. (1985) J. Biol. Chem. 260, 6371-6881], one of the iron ions, Fea, which is easily removed by oxidation to yield the [3Fe-4S]+ cluster of inactive aconitase, shows a dramatic change in the presence of substrate. The remaining iron sites, Feb1,2,3, show minor changes when substrate is bound. Methods devised by us for analyzing and simulating ENDOR spectra of a randomly oriented paramagnet have been used to determine the principal values and orientation relative to the g tensor for the hyperfine tensors of three of the four inequivalent iron sites of the [4Fe-4S]+ cluster, Fea, Feb2, and Feb3, in the substrate-free enzyme and the enzyme-substrate complex. The full tensor for the fourth site, Feb1, could not be obtained because its signal is seen only over a limited range of the EPR envelope. 33S ENDOR data for the enzyme-substrate complex using enzyme reconstituted with 33S show that the four inorganic bridging sulfide ions of the [4Fe-4S]+ cube have isotropic hyperfine couplings of A(S) less than 12 MHz, and analysis indicates that they can be divided into two pairs, one with couplings of A(S1) approximately less than 1 MHz and the other with A(S2) approximately 6-12 MHz; the analysis further places these pairs within the cube relative to the iron sites. 33S data for substrate-free enzyme is qualitatively similar and can be completely simulated by two types of S2- ion, with A(S1) approximately 7.5 and A(S2) approximately 9 MHz; the full hyperfine tensors have been determined. The hyperfine values for the two enzyme forms correspond to surprisingly small unpaired spin density on S2-. 14N ENDOR at Q-band reveals a nitrogen signal that does not change upon substrate binding.     

Wild mouse ecotropic murine leukemia virus infection of inbred mice: dual-tropic virus expression precedes the onset of paralysis and lymphoma.

NFS/N mice inoculated at birth with an ecotropic murine leukemia virus (Cas-Br-MuLV) obtained from wild mice developed hind limb paralysis beginning at 7 weeks of age and nonthymic lymphomas beginning at more than 20 weeks of age. Studies of 1- to 7-week-old Cas-Br-M MuLV-infected mice showed the following: (i) a marked increase in nonecotropic MuLV-related antigens on spleen cells but not thymocytes beginning at 2 weeks; (ii) the appearance of dual-tropic mink cell focus-forming (MCF) MuLV-related gp70 in spleen but not thymus or brain cells at 4 weeks; and (iii) the isolation of infectious MCF MuLV from spleen cells of 7-week-old mice. A role for MCF MuLV in Cas-Br-M MuLV-induced nonthymic lymphomas is indicated by these studies, and a role for recombinant MuLV in neurological disease is considered.     

Electrophoretic Separation of Different Phophosproteins Associated with Ca-ATPase and Na,K-ATPase in Human Red Cell Ghosts

Ca has been found to increase the quantity of ³²P incorporated into red cell ghosts from [γ-³²P]ATP over the levels obtained by incubation with Mg alone or with Mg + Na, in correlation with the effect of Ca on the associated ATPase activities. When the ³²P-labeled ghosts were solubilized in sodium dodecyl sulfate (SDS) and electrophoresed on acrylamide gels only two bands could be detected either by autoradiography or by counting the sliced gels. The faster moving band (P-2) had the same mobility and the same molecular weight (103,000) as the phosphoprotein found either with Mg alone or with Mg + Na. The slower moving band (P-1) was not found in extensively washed ghosts labeled in the absence of Ca. The molecular weight of P-1 is approximately 150,000. P-1 like P-2 was not affected by pretreatment of intact cells with Pronase before labeling indicating that neither the phosphorylating mechanism nor the phosphoprotein are accessible to externally applied Pronase. The demonstration that a Ca-phosphoprotein is separable from the Na-stimulated phosphoprotein suggests that the Ca-ATPase is distinct from and independent of the Na,K-ATPase. The fact that Ca blocks the dephosphorylation by K of the Na-phosphoprotein indicates that caution is required in interpreting results when the activities of the different phosphoproteins have not been separately determined.     

Structural studies of the polysaccharide antigen of Eubacterium saburreum, Strain L 32

The structure of the polysaccharide antigen produced by Eubacterium saburreum, strain L 32, has been investigated. The principal methods used were methylation analysis, graded hydrolysis with acid, and n.m.r. spectroscopy. The polysaccharide, which contains the unusual sugar 3,6-dideoxy-D-arabino-hexose (tyvelose, Tyv), is composed of trisaccharide repeating-units having the following structure:

     

N1-Methyl-2-125I-Lysergic Acid Diethylamide, a Preferred Ligand for In Vitro and In Vivo Characterization of Serotonin Receptors

Methylation of 2-125I-lysergic acid diethylamide (125I-LSD) at the N1 position produces a new derivative, N1-methyl-2-125I-lysergic acid diethylamide (125I-MIL), with improved selectivity and higher affinity for serotonin 5-HT2 receptors. In rat frontal cortex homogenates, specific binding of 125I-MIL represents 80-90% of total binding, and the apparent dissociation constant (KD) for serotonin 5-HT2 receptors is 0.14 nM (using 2 mg of tissue/ml). 125I-MIL also displays a high affinity for serotonin 5-HT1C receptors, with an apparent dissociation constant of 0.41 nM at this site. 125I-MIL exhibits at least 60-fold higher affinity for serotonin 5-HT2 receptors than for other classes of neurotransmitter receptors, with the dopamine D2 receptor as its most potent secondary binding site. Studies of the association and dissociation kinetics of 125I-MIL reveal a strong temperature dependence, with very slow association and dissociation rates at 0 degree C. Autoradiographic experiments confirm the improved specificity of 125I-MIL. Selective labeling of serotonin receptors was observed in all brain areas examined. In vivo binding studies in mice indicate that 125I-MIL is the best serotonin receptor label yet described, with the highest frontal cortex to cerebellum ratio of any serotonergic radioligand. 125I-MIL is a promising ligand for both in vitro and in vivo labeling of serotonin receptors in the mammalian brain.