Toxoplasma gondii: mechanism of resistance to complement-mediated killing

Tachyzoites of the obligate intracellular protozoan Toxoplasma gondii are resistant to lysis in non-immune human serum. We have examined the mechanism of this serum resistance in RH and P strain organisms, which differ markedly in virulence, but are equally resistant to serum killing. Rapid, but limited, activation of the alternative complement pathway occurred in non-immune human serum, with deposition of equivalent amounts of C3 on the two strains. C component C3 bound covalently to parasite acceptor molecules via an ester linkage. The predominant form of C3 was iC3b which cannot participate in formation of a lytic C5b-9 complex. Multiple membrane constituents of the tachyzoite of T. gondii may serve as acceptors for the limited amount of C3 deposited during incubation in non-immune serum. When tachyzoites were presensitized with the lytic anti-p30 mAb 7B8, new amide-linked C3-acceptor complexes formed. Nearly equivalent C3 binding but a threefold enhancement of 125I-C9 binding occurred when mAb 7B8 pre-sensitized tachyzoites were compared to native organisms. These results indicate that tachyzoites of T. gondii are serum resistant because of failure to activate C efficiently. Presensitization with a lytic mAb alters the site of complement deposition and augments C5b-9 formation.     

Proteins derived from platelet alpha granules modulate the uptake of oxidized low density lipoprotein by macrophages.

Activated platelets secrete from their alpha granules a protein-like factor which stimulates the uptake of oxidized low-density lipoprotein (Ox-LDL) by macrophages. The aim of the present study was to evaluate the effect of three purified proteins obtained from platelet alpha granules: platelet-derived growth factor (PDGF), platelet factor-4 (PF-4), and beta-thromboglobulin (B-TG), on the uptake of Ox-LDL by macrophages. Cellular degradation of Ox-LDL by the J-774 A.1 macrophage-like cell line, that was preincubated for 18 h at 37 degrees C, with increasing concentrations of partially purified PDGF, (designated PDGF-CMS-III) was increased by up to 36% in comparison to control cells preincubated without PDGF. This effect was due to PDGF-mediated increase in the number of macrophage receptors for Ox-LDL. The enhanced uptake of Ox-LDL by PDGF resulted in an increase in cellular cholesterol content. Preincubation of macrophages with two types of recombinant PDGF dimers (10 ng/ml), revealed that PDGF-BB stimulated Ox-LDL cellular degradation by 64%, whereas PDGF-AB demonstrated only 34% stimulation, in comparison to control cells that were not treated with PDGF. The stimulatory effect of PDGF-CMS-III and PDGF-AB were reduced by 20% and 28%, respectively, when incubated in the presence of H-7, a specific protein kinase C inhibitor. When macrophages were preincubated with B-TG, cellular uptake of Ox-LDL was reduced by up to 30% at 100 ng B-TG/ml. This effect, however, was obtained only when B-TG was present in the incubation medium. Cellular degradation of Ox-LDL was not affected by preincubation of the cells with PF-4. Pretreatment of PCM with anti-PDGF or anti-B-TG antibodies abolished the effects of PCM on Ox-LDL degradation by macrophages. PDGF, thus, may represent the protein-like factor present in PCM which stimulates Ox-LDL degradation by macrophages, whereas B-TG may have a role in the recognition of PCM particles by the macrophage scavenger receptor. Modulation of macrophage cholesterol content by proteins secreted from activated platelets may have an important role in foam cell formation and atherosclerosis.     

The Regional Distribution of N-Acetylaspartylglutamate (NAAG) and Peptidase Activity Against NAAG in the Rat Nervous System

Abstract: N -Acetylaspartylglutamate (NAAG), a prevalent peptide in the vertebrate nervous system, may be hydrolyzed by extracellular peptidase activity to produce glutamate and N -acetylaspartate. Hydrolysis can be viewed as both inactivating the peptide after synaptic release and increasing synaptic levels of ambient glutamate. To test the hypothesis that NAAG and the peptidase activity that hydrolyzes it coexist as a unique, two-stage system of chemical neurotransmission, 50 discrete regions of the rat CNS were microdissected for assay. In each microregion, the concentration of NAAG was determined by radioimmunoassay and the peptidase activity was assayed using tritiated peptide as substrate. The NAAG concentration ranged from 2.4 nmol/mg of soluble protein in median eminence to 64 in thoracic spinal cord. Peptidase activity against NAAG ranged from 54 pmol of glutamate produced per milligram of membrane protein per minute in median eminence to 148 in superior colliculus. A linear relationship was observed between NAAG peptidase and NAAG concentration in 46 of the 50 areas, with a slope of 2.26 and a correlation coefficient of 0.45. These data support the hypothesis that hydrolysis of NAAG to glutamate and N -acetylaspartate is a consistent aspect of the physiology and metabolism of this peptide after synaptic release. The ratio of peptide concentration to peptidase activity was >0.3 in the following four areas: ventrolateral medulla and reticular formation where the peptide is concentrated in axons of passage, thoracic spinal cord, where NAAG is concentrated in ascending sensory tracts as well as motoneuron cell bodies, and ventroposterior thalamic nucleus.     

Calibrating estimates of phage-induced mortality in marine bacteria: Ultrastructural studies of marine bacteriophage development from one-step growth experiments

The timing of lytic phage development and the relationship between host generation times and latent periods were investigated by electron microscopy of one-step growth experiments in two strains of marine Vibrio species. Results were used in a correction factor developed to interpret field studies of phage-infected marine bacteria. Both the number of mature phage per average cell section and the percentage of cells with mature phage increased exponentially by 73–86% into the latent periods. Assuming that bacterial infection and lysis take place continually in the ocean, conversion factors for relating the percentage of visibly infected bacteria to the total percentage of the bacterial community that are phage-infected were calculated as 3.70–7.14. When this range of factors was applied to previously-collected field data [Proctor LM, Fuhrman JA (1990) Nature (Lond) 343:60–62; Proctor LM, Fuhrman JA (1991) Mar Ecol Prog Ser 69:133–142] from 3 to 31% of the free-living bacteria and 3 to 26% of particulate-associated bacteria appeared to be phage-infected at any given time. Based upon a steady-state model in which half the daughter cells survive to divide again, the percent of total mortality would be twice the total percentage of phage-infected cells. From 6 to 62% and from 6 to 52% of mortality for the free-living and particulate-associated bacterial community, respectively, may be due to viruses. Offprint requests to: L. M. Proctor.     

Phylogenetic diversity of subsurface marine microbial communities from the Atlantic and Pacific Oceans.

The extent of the diversity of marine prokaryotes is not well known, primarily because of poor cultivability. However, new techniques permit the characterization of such organisms without culturing, via 16S rRNA sequences obtained directly from biomass. We performed such an analysis by polymerase chain reaction amplification with universal primers on five oligotrophic open-ocean samples: from 100-m (three samples) and 500-m depths in the western California Current (Pacific Ocean) and from a 10-m depth in the Atlantic Ocean near Bermuda. Of 61 clones, 90% were in clusters of two or more related marine clones obtained by ourselves or others. We report 15 clones related to clone SAR 11 found earlier near Bermuda (S. J. Giovannoni, T. B. Britschgi, C. L. Moyer, and K. G. Field, Nature [London] 345:60-63, 1990), 11 related to marine cyanobacteria, 9 clustered in a group affiliated with gram-positive bacteria, 9 in an archaeal cluster we recently described (mostly from the 500-m sample), 4 in a novel gamma-proteobacterial cluster, and 6 in three two-membered clusters (including other archaea). One clone was related to flavobacteria. Only the cyanobacteria plus one other clone, related to Roseobacter denitrificans (formerly Erythrobacter longus Och114), were within 10% sequence identity to any previously sequenced cultured organism in a major data base. We never found more than two occurrences of the same sequence in a sample, although four times we found identical sequences between samples, two of which were between oceans; one of these sequences was also identical to SAR 11.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of a Small Family (CAIII) of Microsatellite-Containing Sequences with X-Y Homology

Four X-linked loci showing homology with a previously described Y-linked polymorphic locus (DYS413) were identified and characterized. By fluorescent in situ hybridization (FISH), somatic cell hybrids, and YAC screening, the X-linked members of this small family of sequences (CAIII) all map in Xp22, while the Y members map in Yq11. These loci contribute to the overall similarity of the two genomic regions. All of the CAIII loci contain an internal microsatellite of the (CA)_n type. The microsatellites display extensive length polymorphism in two of the X-linked members as well as in the Y members. In addition, common sequence variants are found in the portions flanking the microsatellites in two of the X-linked members. Our results indicate that, during the evolution of this family, length variation on the Y chromosome was accumulated at a rate not slower than that on the X chromosome. Finally, these sequences represent a model system with which to analyze human populations for similar X- and Y-linked polymorphisms. Received: 29 July 1996 / Accepted: 15 January 1997     

Growth efficiencies of freshwater bacterioplankton

The growth efficiency of freshwater bacteria was examined in continuous cultures. One series of experiments was carried out using generation times from 50 to 200 hours and aged, normal, and enriched media, all of natural origin. Another series of experiments examined the bacterial growth efficiency during the growth season in eutrophic Frederiksborg Slotssø, in relation to changes in the planktonic communities and to factors controlling the bacterial incorporation of ³H-thymidine. Attachment of bacteria to the inner surfaces of the experimental flasks was examined using various types of bottles, adding glass tubes to the bottles, and measuring ³H-thymidine incorporation and direct cell counts of attached and free-living bacteria. Attachment of bacteria varied, and in one example up to 36% of the thymidine incorporation was by attached bacteria after 4 days. It was calculated that 36% of attached bacteria caused an underestimation of the growth efficiency of 11%. The mean growth efficiency tended to decrease with generation time using enriched medium (47 to 19%) and aged medium (35 to 12%), and tended to decrease with medium quality (enriched > normal > aged media) from 37% to 27%. The only significant difference in growth efficiency occurred in relation to generation time, in samples with enriched medium (unpaired t-test, P < 0.05). The overall mean value for all generation times and media was 30% (SEM = 3%, n = 24). From April to October, the growth efficiency was determined 5 times in samples from Frederiksborg Slotssø. The overall mean value was 31% (SEM = 3%, n = 30), and there was no significant change in the growth efficiency during the period measured. In June, three bioassay experiments revealed that carbon limitation controlled bacterial incorporation of ³H-thymidine, whereas additions of phosphate and nitrate did not change the incorporation rates. The narrow range of growth efficiencies obtained in this study (mean 31%, SEM = 2%, n = 54) suggests that changes in substratequality in the media applied and in the eutrophic samples examined causes only subtle changes in the growth efficiency.     

Highly efficient bioconversion of glucose into fructose diphosphate with fed-batch-grown Saccharomyces cerevisiae cells

Summary One of the methods commonly used for manufacturing fructose 1,6-diphosphate is based on the enzymatic phosphorylation of glucose with inorganic phosphate using permeabilized brewer's yeast cells. Our results demonstrate that a substantial improvement in the yield of bioconversion can be achieved using fed-batch-grown Saccharomyces cerevisiae cells. Under an appropriate glucose and phosphate to cell ratio the efficiency of bioconversion reaches 70% of the theoretical value. Offprint requests to: C. Compagno     

DNA hybridization to compare species compositions of natural bacterioplankton assemblages.

Little is known about the species composition and variability of natural bacterial communities, mostly because conventional identification requires pure cultures, but less than 1% of active natural bacteria are cultivable. This problem was circumvented by comparing species compositions via hybridization of total DNA of natural bacterioplankton communities for the estimation of the fraction of DNA in common between two samples (similarity). DNA probes that were labeled with 35S by nick translation were hybridized to filter-bound DNA in a reciprocal fashion; similarities (in percent) were calculated by normalizing the values to self-hybridizations. In tests with DNA mixtures of pure cultures, the experimentally observed similarities agreed with expectations. However, reciprocal similarities (probe and target reversed) were often asymmetric, unlike those of DNA from single strains. This was due to the relative complexity and G + C content of DNA, which provided a means to interpret the asymmetry that was occasionally observed in natural samples. Natural bacteria were collected by filtration from Long Island Sound (LIS), N.Y., the Caribbean and Sargasso seas, and a coral reef lagoon near Bermuda. The samples showed similarities of less than 10 to 95%. The LIS and Sargasso and Caribbean sea samples were 20 to 50% similar to each other. The coral reef sample was less than 10% similar to the others, indicating its unique composition. Seasonality was also observed; an LIS sample obtained in the autumn was 40% similar to two LIS samples obtained in the summer; these latter two samples were 95% similar. We concluded that total DNA hybridization is a rapid, simple, and unbiased method for investigating the variation of bacterioplankton species composition over time and space, avoiding the need of culturing.