Enhanced resistance of anaerobic rumen fungi to the ionophores monensin and lasalocid in the presence of methanogenic bacteria

The presence of Methanobrevibacter smithii altered the susceptibility of the anaerobic fungi Neocallimastix frontalis and Piromonas communis to the carboxylic ionophores monensin and lasalocid. The ionophores depressed growth (measured by chitin accretion), the uptake of glucose and the production of H2, formate and acetate by the fungi growing axenically in semi-solid medium. In the presence of M. smithii , the sensitivity of the fungi to monensin and lasalocid was decreased. For example, the uptake of glucose by N. frontalis strain RE1 in the culture was reduced to 50% of the control value by monensin at 0.5 mUg/ml. In the presence of M. smithii strain PS, approximately three tunes as much monensin was needed to bring about the same effect. In similar tests, the sensitivity of strain RE1 to lasalocid was decreased about nine-fold in the presence of M. smithii. The effect was not observed if the methanogens were killed by autoclaving before inoculation. It is suggested that the enhanced resistance to ionophores in the presence of M. smithii is a consequence of changes in the energy metabolism of the fungi growing in co-culture.     

Work capacity during 30 days of bed rest with isotonic and isokinetic exercise training

The purpose was to test the hypothesis that twice daily, short-term, variable intensity isotonic and intermittent high-intensity isokinetic leg exercise would maintain peak O2 uptake (VO2) and muscular strength and endurance, respectively, at or near ambulatory control levels during 30 days of -6 degrees head-down bed rest (BR) deconditioning. Nineteen men (aged 32-42 yr) were divided into no exercise control (peak VO2 once/wk, n = 5), isokinetic (Lido ergometer, n = 7), and isotonic (Quinton ergometer, n = 7) groups. Exercise training was conducted in the supine position for two 30-min periods/day for 5 days/wk. Isotonic training was at 60-90% of peak VO2, and isokinetic training (knee flexion-extension) was at 100 degrees/s. Mean (+/- SE) changes (P less than 0.05) in peak VO2 (ml.m-1.kg-1) from ambulatory control to BR day 28 were 44 +/- 4 to 36 +/- 3, -18.2% (3.27-2.60 l/m) for no exercise, 39 +/- 4 to 40 +/- 3, +2.6% (3.13-3.14 l/min) for isotonic, and 44 +/- 3 to 40 +/- 2, -9.1% (3.24-2.90 l/min) for isokinetic. There were no significant changes in any groups in leg peak torque (right knee flexion or extension), leg mean total work, arm total peak torque, or arm mean total work. Mean energy costs for the isotonic and isokinetic exercise training were 446 kcal/h (18.8 +/- 1.6 ml.min-1.kg-1) and 214 kcal/h (8.9 +/- 0.5 ml.m-1.kg-1), respectively. Thus near-peak, variable intensity, isotonic leg exercise maintains peak VO2 during 30 days of BR, while this peak, intermittent, isokinetic leg exercise protocol does not.     

Characterization of proteins secreted by sheep oviduct epithelial cells and their function in embryonic development

The role in early development of proteins secreted by oviduct epithelial cells has been investigated. Secreted proteins devoid of serum contamination have been produced by the surgical removal and immediate incubation of oviduct cells in [35S]methionine-containing medium. After electrophoretic separation, secreted polypeptides could be divided into those that were secreted uniformly throughout the oestrous cycle and a second class that showed a cyclical pattern of secretion. The first class of proteins represented a small proportion of total output whilst the predominant second class was composed mainly of polypeptides of Mr 92 and 46 x 10(3), respectively. Both of these polypeptide species, referred to as sheep oviduct proteins 92 and 46 (SOP 92, SOP 46), are detected only during the first 4 to 5 days after oestrus when the embryos are located in the oviduct. Oviduct cells collected at oestrus and maintained thereafter in culture secrete the same pattern of proteins and follow the same time course as their counterparts in vivo. The interaction between the oviduct proteins and the developing embryo was studied firstly by determining whether any of the secreted proteins bound to the zona pellucida. The results of iodination studies showed that two polypeptides of Mr 92 and 46 x 10(3), respectively, were bound to the zona pellucida of eggs removed from the oviduct but were absent from eggs that had not had contact with the oviduct epithelium. That these newly acquired proteins represent SOP 92 and 46 is suggested by their electrophoretic mobility and their ability to bind to the zona of follicular eggs when added in vitro and by the fact that they both disappear from the zonae of embryos after exit from the oviduct. The collection of unlabelled secreted proteins enabled us to produce a monoclonal antibody, which was used in the second series of experiments on oviduct-embryo interactions. The results confirmed that SOP 92 binds to the zona pellucida and moreover showed that this protein crosses the zona and becomes associated with the individual blastomeres of the developing embryo. These findings provide evidence that the mammalian oviduct probably plays a direct role in supporting embryonic development through specific polypeptides produced by its epithelium.     

PDGF receptors on cells of the oligodendrocyte-type-2 astrocyte (O-2A) cell lineage

It has been shown previously that cultures of rat optic nerve contain three types of macroglial cells--oligodendrocytes and two types of astrocytes. Type-1 astrocytes develop from their own precursor cells beginning before birth, while oligodendrocytes and type-2 astrocytes develop postnatally from a common bipotential precursor called the O-2A progenitor cell. Proliferating O-2A progenitor cells give rise to postmitotic oligodendrocytes beginning around birth, and to type-2 astrocytes beginning in the second postnatal week. Studies in vitro have suggested that platelet-derived growth factor (PDGF), secreted by type-1 astrocytes, plays an important part in timing oligodendrocyte development: PDGF seems to keep O-2A progenitor cells proliferating until an intrinsic clock in the progenitor cells initiates the process leading to oligodendrocyte differentiation. The clock apparently determines when a progenitor cell becomes unresponsive to PDGF, at which point the cell stops dividing and, as a consequence, automatically differentiates into an oligodendrocyte. Here we have used radiolabelled PDGF to show that O-2A progenitor cells have PDGF receptors, suggesting that these cells respond directly to PDGF. The receptors resemble the type A PDGF receptor previously described on human fibroblasts and are initially retained when progenitor cells stop dividing and develop in vitro into oligodendrocytes. The latter finding indicates that receptor loss is not the reason that progenitor cells initially become mitotically unresponsive to PDGF.     

Both A1 and A2a Purine Receptors Regulate Striatal Acetylcholine Release

The receptors responsible for the adenosine-mediated control of acetylcholine release from immunoaffinity-purified rat striatal cholinergic nerve terminals have been characterized. The relative affinities of three analogues for the inhibitory receptor were (R)-phenylisopropyladenosine greater than cyclohexyladenosine greater than N-ethylcarboxamidoadenosine (NECA), with binding being dependent of the presence of Mg2+ and inhibited by 5'-guanylylimidodiphosphate [Gpp(NH)p] and adenosine receptor antagonists. Adenosine A1 receptor agonists inhibited forskolin-stimulated cholinergic adenylate cyclase activity, with an IC50 of 0.5 nM for (R)-phenylisopropyladenosine and 500 nM for (S)-phenylisopropyladenosine. A1 agonists inhibited acetylcholine release at concentrations approximately 10% of those required to inhibit the cholinergic adenylate cyclase. High concentrations (1 microM) of adenosine A1 agonists were less effective in inhibiting both adenylate cyclase and acetylcholine release, due to the presence of a lower affinity stimulatory A2 receptor. Blockade of the A1 receptor with 8-cyclopentyl-1,3-dipropylxanthine revealed a half-maximal stimulation by NECA of the adenylate cyclase at 10 nM, and of acetylcholine release at approximately 100 nM. NECA-stimulated adenylate cyclase activity copurified with choline acetyltransferase in the preparation of the cholinergic nerve terminals, suggesting that the striatal A2 receptor is localized to cholinergic neurones. The possible role of feedback inhibitory and stimulatory receptors on cholinergic nerve terminals is discussed.     

In vivo cellular tropism of human T-cell leukemia virus type 1.

To establish the phenotype of human T-cell leukemia virus type 1 (HTLV-1)-infected cells in peripheral blood, the polymerase chain reaction was used to detect and quantitate viral DNA in subpopulations of leukocytes obtained from patients with tropical spastic paraparesis and asymptomatic carriers. HTLV-1 could not be detected in peripheral blood mononuclear cells thoroughly depleted of T lymphocytes (E- CD3-), nor could it be detected in highly enriched populations of B lymphocytes (E- CD19+), monocytes (E- CD14+), or natural killer cells (E- CD16+). T lymphocytes were strongly positive for HTLV-1, and fractionation of this population revealed that 90 to 99% of the HTLV-1 DNA segregated with the CD4+ CD8- and CD45RO+ subsets. No difference between the cell type distribution of HTLV-1 in the asymptomatic carrier and the subjects with tropical spastic paraparesis was evident. Southern hybridization of genomic DNA prepared from the peripheral blood of HTLV-1 carriers indicated that up to 10% of circulating leukocytes may carry the HTLV-1 provirus.     

Human immunodeficiency virus pseudotypes with expanded cellular and species tropism.

One mechanism for expanding the cellular tropism of a virus is through the formation of phenotypically mixed particles or pseudotypes, a process commonly occurring during viral assembly in cells infected with two or more viruses. We report here that dual infection of cells with human immunodeficiency virus (HIV) and a murine amphotropic retrovirus leads to the production of HIV pseudotypes that have acquired the host range of the amphotropic retrovirus and are capable of infecting not only CD4- human cells but also mouse cells. The replication of the HIV pseudotypes in the various CD4- cells was determined by measuring the appearance of HIV antigens in the supernatants, by cocultivation of CD4+ CEM cells with the infected CD4- cells, and in some cases by assaying the culture supernatants directly for infectious virus. Of the cells tested, human foreskin fibroblasts were the best host cells, and by in situ cytohybridization, we were able to document that all cells in the culture were infected. In addition, the temporal appearance of HIV-specific proteins in the HIV pseudotype-infected fibroblasts was similar to that seen in CD4+ CEM cells. If the human fibroblasts were first infected with the amphotropic retrovirus, they demonstrated the property of superinfection exclusion and were resistant to subsequent infection by the HIV pseudotype. In other cell lines, including the human glioblastoma-derived cell line U373MG, HeLa cells, BALB/c mouse embryo cells, and SC-1 wild mouse cells, although the HIV pseudotype infection appeared to be less efficient, substantial amounts of HIV were nevertheless produced. These results indicate that the HIV (amphotropic retrovirus) pseudotypes may be useful for studying the molecular biology of HIV infections in a wide range of cells.     

Functional role of human IgG subclasses in eosinophil-mediated killing of schistosomula of Schistosoma mansoni

Although IgG antibodies and eosinophils have been shown to kill schistosomula of Schistosoma mansoni in vitro, very little data exist that describe the role of each IgG antibody isotype in this event. This study was designed to test the role of each IgG subclass in the eosinophil-dependent killing reaction. IgG antibodies purified by protein G or protein A affinity chromatography demonstrated a killing effect only in the presence of eosinophils activated in vivo or normal eosinophils activated in vitro by eosinophil activating factor. Purification of each IgG isotype allowed confirmation of these results and demonstrated that the killing effect was associated with IgG1 and IgG3 antibodies. IgG2 antibodies expressed a dual function: 1) an effector function with activated eosinophils and 2) a blocking function with normal eosinophils. IgG4 antibodies, whatever the source of eosinophils, blocked the killing mediated by IgG effector antibodies. These findings are discussed in relation to immunity and susceptibility to reinfection in human schistosomiasis.     

Role of acidic intracellular compartments in the biosynthesis of Dictyostelium lysosomal enzymes. The weak bases ammonium chloride and chloroquine differentially affect proteolytic processing and sorting

Radiolabel pulse-chase and subcellular fractionation procedures were used to analyze the transport, proteolytic processing, and sorting of two lysosomal enzymes in Dictyostelium discoideum cells treated with the weak bases ammonium chloride and chloroquine. Dictyostelium lacks detectable cation-independent mannose-6-phosphate receptors and represents an excellent system to investigate alternative mechanisms for lysosomal enzyme targeting. Exposure of growing cells to ammonium chloride, which increased the pH in intracellular vacuoles from 5.4 to 5.8-6.1, slowed but did not prevent the proteolytic processing and correct localization of pulse-radiolabeled precursors to the lysosomal enzymes alpha-mannosidase and beta-glucosidase. Additionally, ammonium chloride did not affect transport of the enzymes to the Golgi complex, as they acquired resistance to the enzyme endoglycosidase H at the same rate as in control cells. When the pH of lysosomal and endosomal organelles was raised to 6.4 with higher concentrations of ammonium chloride, the percentage of secreted (apparently mis-sorted) precursor polypeptides increased slightly, but proteolytic processing of intermediate forms of lysosomal enzymes to mature forms was greatly reduced. The intermediate and mature forms of alpha-mannosidase and beta-glucosidase did, however, accumulate intracellularly in vesicles similar in density to lysosomes. In contrast, in cells exposed to low concentrations of chloroquine the intravacuolar pH increased only slightly (to 5.7); however, enzymes were inefficiently processed and, instead, rapidly secreted as precursor molecules. Experiments involving the addition of chloroquine at various times during the chase of pulse-radiolabeled cells demonstrated that this weak base acted on a distal Golgi or prelysosomal compartment to prevent the normal sorting of lysosomal enzymes. These results suggest that although acidic endosomal/lysosomal compartments may be important for the complete proteolytic processing of lysosomal enzymes in Dictyostelium, low pH is not essential for the proper targeting of precursor polypeptides. Furthermore, certain amines may induce mis-sorting of these enzymes by pH-independent mechanisms.     

Formation of O6-methyldeoxyguanosine at specific sites in a synthetic oligonucleotide designed to resemble a known mutagenic hotspot

Four synthetic oligodeoxyribonucleotides of the sequence 5'-CCG1TG2G3G4ATATGGGCTG-3' were constructed with a 1',2'-[3H]deoxyguanosine located at one of the four sites indicated (1, 2, 3, or 4). This sequence was derived from a region of the Escherichia coli xanthine-guanine phosphoribosyltransferase gene where position 4 is a site frequently mutated by N-methyl-N'-nitrosourea as compared to sites 1-3. These four oligomers were alkylated in both single- and double-stranded form with N-methyl-N'-nitrosourea, and the relative amount of O6-methyldeoxyguanosine (O6-MedGuo) formed at each position was quantitated. Up to a 5-6-fold greater formation of O6-MedGuo was observed at positions 3 and 4 as compared to positions 1 and 2. This uneven distribution was only observed in oligomers in the double-stranded form, suggesting that secondary structure was an important determinant in generating the uneven distribution of O6-MedGuo. Comparisons between the extent of O6-MedGuo formation and mutation frequency at the four positions suggest that a difference in the formation of promutagenic adducts at specific sites is just one of the factors involved in the generation of mutagenic "hotspots." The novel method developed was applied to the study of formation of O6-MedGuo at specific sites; however, it should be suitable for studying the formation and repair of DNA adducts generated by a variety of chemicals in a wide variety of DNA sequences.