The effects of antibodies and complement in macrophage-mediated cytotoxicity on metacercariae of the lung fluke, Paragonimus westermani

Paragonimus westermani is a tissue migrating parasite in the early stage until arriving at lung, and most of the parasites spend their life spans there. Considerable immune responses including activation of macrophages are taken place during the residence of parasites in the host. However, concerning the immunologic defense mechanisms of the host against this parasite, only a few document is available so far. In this study, the cytotoxic effect of peritoneal macrophages under the presence of antibody and/or complement against metacercariae of P. westermani was investigated in vitro. Metacercariae were collected from the crayfish, Cambaroides similis and hatched out in Tyrode solution (pH 7.4). Plastic adherent cells from normal or infected rat (Wistar) peritoneal exudates were used as experimental macrophages. Polyclonal antibodies were obtained from infected rats and a cat. Cat IgG was fractioned with ion exchange chromatography. Fresh rabbit complement was used according to experimental scheme. Various combinations of peritoneal macrophages, normal or infected rat serum, complement and cat IgG were incubated at 36 degrees C in 5% CO2 incubator for 6, 14, 24 and 48 hours. The results obtained were as follows: 1. P. westermani infection activated peritoneal macrophages non-specifically and this activation induced increases of cell adherence and cytotoxicity on metacercariae. 2. In the presence of infected rat serum the antibody-dependent cell-mediated cytotoxicity of peritoneal macrophages on metacercariae was significantly increased and showed a peak at 6-hour incubation. But the cytotoxic effect was markedly reduced after inactivation of complement and heat-labile IgE antibody by the heating of infected serum at 56 degrees C for 30 minutes. 3. The highest cytotoxic effect (100%) of concomitant incubation with IgG and complement showed 24 hours after incubation, although cell adherence was relatively low at 6-hour incubation and 0% at 24-hour incubation. 4. Coordinative functions of complement with serum and IgG were effective in cell adherence and in cytotoxicity, but it is not clear the independent role of complement on the macrophage-mediated cytotoxicity in this study. With these results it is assumed that P. westermani infection can induce the non-specific activation of peritoneal macrophages, and serum antibodies including IgE antibody might enhance the cytotoxicity by macrophages.     

Maturation-associated loss and incomplete de novo synthesis of the transferrin receptor in peripheral sheep reticulocytes: response to heme and iron

Hemin, but not iron, in the culture medium stimulates the maturation-associated loss of the transferrin receptor from sheep reticulocytes (t1/2 for loss approximately 6 hr) and its appearance in a population of externalized vesicles. A similar pattern is seen with nucleoside binding (a measure of the nucleoside transporter), where hemin increases the loss of binding activity from the cells during culture, concomitant with an increase in nucleoside binding in the externalized vesicles. Sheep reticulocytes retain the ability to synthesize the transferrin receptor, but the 35S-labeled receptors are not detected in released vesicles. Whereas hemin stimulates the loss of 35S-labeled transferrin receptors from the cell (t1/2 for loss approximately 20 hr), nonheme iron is more effective than heme. This difference in response of native and 35S-labeled receptor to hemin and iron supplements appears to be related to the differences in the two classes of receptors. Although the 35S-labeled receptor binds transferrin and both native and 35S-labeled peptides comigrate after chemical deglycosylation, the 35S-receptor is approximately 2 kD smaller than the native receptor and fails to acquire its complete size even when chased for up to 24 hr. Moreover, the 35S-labeled receptor is not expressed at the cell surface, but is retained in a nonrecycling compartment, where it is insensitive to digestion by trypsin at both 0 degrees C and 37 degrees C.     

Sites of lymph follicle formation in the draining popliteal lymph nodes of mice locally injected with antigenic and mitogenic substances

Our previous studies showed that some antigenic and mitogenic substances, when locally injected into mice, efficiently produced new lymph follicles outside pre-existing follicles in draining lymph nodes, whereas others had virtually no effect. In the present experiments, young adult male mice were injected with several antigens and mitogens in the rear footpad, and the number and development sites of newly produced lymph follicles in the draining popliteal nodes were studied using serial sections of the nodes obtained between 5 and 21 days after injection. In the unstimulated state, each popliteal node contained a limited number of lymph follicles which mostly lay in a portion of the peripheral cortex overlaying the deep cortex (this portion is referred to as the PCOU), whereas a portion of the peripheral cortex extending beyond the deep cortex (referred to as the PCBU) was underdeveloped with only occasional follicles. Mice treated with soluble PHA or fluid tetanus toxoid developed germinal centers in association with existing follicles but failed to produce new follicles. The PCBU of the draining nodes remained underdeveloped, and the number and distribution pattern of lymph follicles within a draining node were comparable to those in the control node. Animals treated with LPS (50 micrograms), Con A, alum-precipitated PHA or alum-precipitated tetanus toxoid produced significantly large numbers of new follicles outside pre-existing follicles in the draining nodes, the new follicles produced in the PCBU being generally more numerous than those in the PCOU. In these draining nodes, the peripheral cortex, comprising a number of follicles, was found to overlie the deep cortex and extend beyond the deep cortex towards the hilar region. In animals given a less effective stimulant, such as ferritin or a smaller dose of LPS (10 micrograms), the draining nodes produced a relatively small number of new follicles, most of which were formed in the PCBU. The present results indicate that in the mouse popliteal node, the PCBU is morphologically underdeveloped under normal conditions, but develops lymph follicles in response to exogenous stimuli more readily than the PCOU, and that substances efficient in inducing follicle formation can be regarded as capable of stimulating the development of the peripheral cortex.     

Use of electrophoretic enzyme patterns for streptomycete systematics

Abstract The relative electrophoretic mobilities of various enzymes from 24 different streptomycetes were determined on polyacrylamide gels in order to examine the relatedness of species and strains of the genus Streptomyces . Of 11 different enzymes tested in this study, hexokinase, glucose-6-phosphate dehydrogenase, phosphogluconate dehydrogenase, malate dehydrogenase and isocitrate dehydrogenase showed a limited number of constant and reproducible polymorphic enzyme patterns, by comparing which the inter-specific relationships could be examined. In contrast, glucose dehydrogenase, alcohol dehydrogenase, 3-hydroxybutyrate dehydrogenase, phosphoglucose isomerase, peroxidase and esterase exhibited either weak non-reproducible or highly heterogeneous band patterns which were suitable for dissecting the strains within a species and a cluster group.     

Saturated Molecular Map of the Rice Genome Based on an Interspecific Backcross Population

A molecular map has been constructed for the rice genome comprised of 726 markers (mainly restriction fragment length polymorphisms; RFLPs). The mapping population was derived from a backcross between cultivated rice, Oryza sativa, and its wild African relative, Oryza longistaminata. The very high level of polymorphism between these species, combined with the use of polymerase chain reaction-amplified cDNA libraries, contributed to mapping efficiency. A subset of the probes used in this study was previously used to construct an RFLP map derived from an inter subspecific cross, providing a basis for comparison of the two maps and of the relative mapping efficiencies in the two crosses. In addition to the previously described PstI genomic rice library, three cDNA libraries from rice (Oryza), oat (Avena) and barley (Hordeum) were used in this mapping project. Levels of polymorphism detected by each and the frequency of identifying heterologous sequences for use in rice mapping are discussed. Though strong reproductive barriers isolate O. sativa from O. longistaminata, the percentage of markers showing distorted segregation in this backcross population was not significantly different than that observed in an intraspecific F(2) population previously used for mapping. The map contains 1491 cM with an average interval size of 4.0 cM on the framework map, and 2.0 cM overall. A total of 238 markers from the previously described PstI genomic rice library, 250 markers from a cDNA library of rice (Oryza), 112 cDNA markers from oat (Avena), and 20 cDNA markers from a barley (Hordeum) library, two genomic clones from maize (Zea), 11 microsatellite markers, three telomere markers, eleven isozymes, 26 cloned genes, six RAPD, and 47 mutant phenotypes were used in this mapping project. Applications of a molecular map for plant improvement are discussed.     

Expression and secretion of human lipocortin-1 by promoter and signal sequence of STA1 from Saccharomyces diastaticus

Summary For the secretion of human lipocortin-1 (LC-1) in yeast, a expression and secretion vector was constructed by using the promoter and signal sequence of glucoamylase gene (STA1) of Saccharomyces diastaticus. After the cDNA of human LC-1 was ligated with the secretion vector, the resulting hybrid plasmid was transformed into S. diastaticus. When the recombinant S. diastaticus was cultivated in YPD medium, LC-1 was expressed and secreted into the extracellular medium, yielding LC-1 protein at a concentration of 2.5 g/mL.     

Homoeologous relationships of rice, wheat and maize chromosomes

A set of cDNA clones, which had previously been mapped onto wheat chromosomes, was genetically mapped onto the chromosomes of rice. The resulting comparative maps make it possible to estimate the degree of linkage conservation between these two species. A number of chromosomal rearrangements, some of which must have involved interchromosomal translocations, differentiate the rice and wheat genomes. However, synteny of a large proportion of the loci appears to be conserved between the two species. The results of this study, combined with those from a recently published comparative map of the rice and maize genomes, suggest that rice, wheat and maize share extensive homoeologies in a number of regions in their genomes. Some chromosomes (e.g. chromosome 4 in rice, chromosomes 2 and 2S in wheat and maize, respectively) may have escaped major rearrangement since the divergence of these species from their last common ancestor. Comparative maps for rice, wheat and maize should make it possible to begin uniting the genetics of these species and allow for transfer of mapping information (including centromere positions) and molecular marker resources (e.g. RFLP probes) between species. In addition, such maps should shed light on the nature of chromosome evolution that accompanied the radiation of grasses in the early stages of plant diversification.     

Metabolic engineering of Mannheimia succiniciproducens for malic acid production using dimethylsulfoxide as an electron acceptor

Microbial production of various TCA intermediates and related chemicals through the reductive TCA cycle has been of great interest. However, rumen bacteria that naturally possess strong reductive TCA cycle have been rarely studied to produce these chemicals, except for succinic acid, due to their dependence on fumarate reduction to transport electrons for ATP synthesis. In this study, malic acid (MA), a dicarboxylic acid of industrial importance, was selected as a target chemical for mass production using Mannheimia succiniciproducens, a rumen bacterium possessing a strong reductive branch of the TCA cycle. The metabolic pathway was reconstructed by eliminating fumarase to prevent MA conversion to fumarate. The respiration system of M. succiniciproducens was reconstructed by introducing the Actinobacillus succinogenes dimethylsulfoxide (DMSO) reductase to improve cell growth using DMSO as an electron acceptor. Also, the cell membrane was engineered by employing Pseudomonas aeruginosa cis-trans isomerase to enhance MA tolerance. High inoculum fed-batch fermentation of the final engineered strain produced 61 g/L of MA with an overall productivity of 2.27 g/L/h, which is the highest MA productivity reported to date. The systems metabolic engineering strategies reported in this study will be useful for developing anaerobic bioprocesses for the production of various industrially important chemicals.     

Reductive Dehalogenation of Brominated Phenolic Compounds by Microorganisms Associated with the Marine Sponge Aplysina aerophoba

Marine sponges are natural sources of brominated organic compounds, including bromoindoles, bromophenols, and bromopyrroles, that may comprise up to 12% of the sponge dry weight. Aplysina aerophoba sponges harbor large numbers of bacteria that can amount to 40% of the biomass of the animal. We postulated that there might be mechanisms for microbially mediated degradation of these halogenated chemicals within the sponges. The capability of anaerobic microorganisms associated with the marine sponge to transform haloaromatic compounds was tested under different electron-accepting conditions (i.e., denitrifying, sulfidogenic, and methanogenic). We observed dehalogenation activity of sponge-associated microorganisms with various haloaromatics. 2-Bromo-, 3-bromo-, 4-bromo-, 2,6-dibromo-, and 2,4,6-tribromophenol, and 3,5-dibromo-4-hydroxybenzoate were reductively debrominated under methanogenic and sulfidogenic conditions with no activity observed in the presence of nitrate. Monochlorinated phenols were not transformed over a period of 1 year. Debromination of 2,4,6-tribromophenol, and 2,6-dibromophenol to 2-bromophenol was more rapid than the debromination of the monobrominated phenols. Ampicillin and chloramphenicol inhibited activity, suggesting that dehalogenation was mediated by bacteria. Characterization of the debrominating methanogenic consortia by using terminal restriction fragment length polymorphism (TRFLP) and denaturing gradient gel electrophoresis analysis indicated that different 16S ribosomal DNA (rDNA) phylotypes were enriched on the different halogenated substrates. Sponge-associated microorganisms enriched on organobromine compounds had distinct 16S rDNA TRFLP patterns and were most closely related to the δ subgroup of the proteobacteria. The presence of homologous reductive dehalogenase gene motifs in the sponge-associated microorganisms suggested that reductive dehalogenation might be coupled to dehalorespiration.     

Effect of Root Zone Temperature on the Mineral Composition of Xylem Sap and Plasma Membrane K+-Mg++-ATPase Activity of Grafted-Cucumber and -Figleaf Gourd Root Systems

Cucumber (Cucumis sativus L.) seedlings were grafted onto cucumber-(CG) or figleaf gourd- (FG, Cucurbita ficifolia Bouché)seedlings in order to determine the effect of solution temperature(12, 22, and 32°C) on the mineral composition of xylem sapand the plasma membrane K⁺-Mg⁺⁺-ATPase activities of the roots.Low solution temperature (12°C) lowered the concentrationof NO^–₃ and H₂PO^–₄ in xylem sap of CG plants butnot of FG plants. Concentrations of K⁺, Ca⁺⁺ and Mg⁺⁺ in xylemsap were less affected than anions by solution temperature.The plasma membrane of FG plants grown in 12°C solutiontemperature showed the highest K⁺- Mg⁺⁺-ATPase activity at allATP concentrations up to 3 mM and at low reaction temperatureup to 12°C, indicating resistance of figleaf gourd to lowroot temperature. (Received December 27, 1994; Accepted March 10, 1995)