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Biomedical subjects

D F Niven

Publications and source records attributed to D F Niven.

At least 19 recordsLinked to original sources

Apolipophorin-III in the greater wax moth, Galleria mellonella (Lepidoptera: Pyralidae).

The level of apolipophorin-III reached a maximum in the haemolymph of Galleria mellonella at the end of the feeding phase of the seventh larval instar and declined to a plateau value in the pupal and the adult stages. Apolipophorin-III was detected immunologically in fat body tissue, haemocyte lysates, and plasma. In its native state, apolipophorin-III may be associated with another protein with an apparent molecular mass of 77 kDa, possibly apolipophorin-II. Injections of octopamine did not cause lipid loading of high density lipophorin.

Animals↗

Production of transferrin receptors by Histophilus ovis: three of five strains require two signals.

Five strains of Histophilus ovis (9L, 642A, 714, 5688T, and 3384Y) were investigated with respect to iron acquisition. All strains used ovine, bovine, and goat transferrins (Tfs), but not porcine or human Tfs, as iron sources for growth. In solid phase binding assays, total membranes from only two (9L and 642A) of the five strains, grown under iron-restricted conditions, were able to bind Tfs (ovine, bovine, and goat, but not porcine or human). However, when the organisms were grown under iron-restricted conditions in the presence of bovine transferrin (Tf), total membranes from all strains exhibited Tf binding (as above); competition experiments demonstrated that all three Tfs (ovine, bovine, and goat) were bound by the same receptor(s). Membranes from organisms grown under iron-replete conditions in the presence or absence of bovine Tf failed to bind any of the test Tfs. An affinity-isolation procedure allowed the isolation of two putative Tf-binding polypeptides (78 and 66 kDa) from total membranes of strains 9L and 642A grown under iron-restricted conditions, and from membranes of all strains if the growth medium also contained Tf. It is concluded that all strains tested acquire Tf-bound iron by means of siderophore-independent mechanisms involving surface receptors analogous to the Tf-binding proteins (TbpA and TbpB) found in comparable organisms; although iron restriction alone is sufficient to promote the expression of these proteins by strains 9L and 642A, their production by strains 714, 5688T, and 3384Y appears to require two signals, iron restriction and the presence of Tf.

Animals↗

Iron content of Streptococcus suis and evidence for a dpr homologue.

The type strain of Streptococcus suis was investigated for features that might help the organism to tolerate the H2O2 that is produced during growth. Enzyme assays, using soluble extracts, revealed that the type strain, which lacks catalase, lacks NADH peroxidase in both the mid-exponential and stationary phases of the growth cycle. Although iron could not be detected colourimetrically in dense cell suspensions, determination of the cellular iron content following growth to early stationary phase in the presence of 55FeCl3 demonstrated that S. suis does contain iron and hence is incapable of iron exclusion. Gene amplification, using oligonucleotide primers based on dpr of Streptococcus mutans, followed by nucleotide sequencing, revealed in S. suis, the presence of a gene that encodes a Dpr homologue. It is concluded that in S. suis, tolerance of H2O2 is due to iron sequestration by Dpr and the consequent effect of this process on the extent of Fenton chemistry.

Bacterial Proteins↗

Effects of Plagiorchis elegans (Trematoda: Plagiorchiidae) infection on the carbohydrate metabolism of fourth instar Aedes aegypti (Diptera: Culicidae).

Infection of fourth-instar Aedes aegypti (L.) with the entomopathogenic digenean Plagiorchis elegans (Rudolphi) alters the carbohydrate metabolism of the insect. Within 24 h of cercarial penetration, total body extracts of infected fourth instars exhibited decreased trehalase activity, increased trehalose-6-phosphatase activity, and a concomitant accumulation of trehalose when compared with uninfected larvae. The amounts of glucose, glycogen and lipids, and the activity of glycogen phosphorylase a were similar in extracts of infected and control larvae. The predominant fatty acids, in both control and infected larvae, were C 18:0, C 18:1, and C 18:3. There were no significant differences in the types or proportions of fatty acids found in control and infected larvae. Parasitic infection is discussed in terms of impaired trehalose metabolism.

Aedes↗

Apolipophorin-III and the interactions of lipoteichoic acids with the immediate immune responses of Galleria mellonella.

We investigated the effects of lipoteichoic acids, surface components of Gram-positive bacteria, on the hemocytes and phenoloxidase activity in last instar Galleria mellonella larvae, as well as the binding of apolipophorin-III, an insect lipid-binding protein, to lipoteichoic acids. Binding of apolipophorin-III to lipoteichoic acid was studied using an assay based on 1,9-dimethylmethylene blue. Apolipophorin-III bound the lipoteichoic acids from Bacillus subtilis, Enterococcus hirae, and Streptococcus pyogenes and to intact cells of E. hirae. E. hirae lipoteichoic acid promoted the binding of apolipophorin-III to the cells of this species. All lipoteichoic acids tested caused a dose- and time-dependent drop in the total counts of hemocytes and, depending on the species of lipoteichoic acid, partial or complete depletion of plasmatocytes. Granulocyte counts were not affected. Apolipophorin-III prevented partially the loss of plasmatocytes due to B. subtilis lipoteichoic acid. All three lipoteichoic acids studied activated phenoloxidase in vitro; injections of B. subtilis lipoteichoic acid into the larvae elevated the phenoloxidase activity, whereas injections of E. hirae or S. pyogenes lipoteichoic acid, or apolipophorin-III alone, suppressed it. Apolipophorin-III decreased the activation of phenoloxidase by B. subtilis lipoteichoic acid.

Animals↗

Effects of iron and manganese availability on growth and production of superoxide dismutase by Streptococcus suis.

A complex medium supported good growth of the type strain of Streptococcus suis irrespective of the presence or absence of a high concentration (1 microM) of the iron chelating agent, ethylenediamine di-o-hydroxyphenylacetic acid. Good growth was also obtained using a complex medium that had been treated with Chelex-100 to reduce the iron content, but only if this medium was supplemented with manganese; supplementation with iron had little effect. Collectively, these results indicate that S. suis requires manganese, but not iron, for growth. Polyacrylamide gel electrophoresis of cell extracts followed by activity staining revealed the presence of a single manganese-cofactored superoxide dismutase; activity staining and enzyme assays revealed that manganese availability during growth affected the activity of the superoxide dismutase in cell extracts. The results are discussed with respect to the capacity of S. suis to avoid damage by reactive oxygen species.

Iron↗

Comparative study of iron acquisition by biotype 1 and biotype 2 strains of Actinobacillus pleuropneumoniae.

Four strains of the swine pathogen, Actinobacillus pleuropneumoniae, namely, the type strain (ATCC 27088; biotype 1), the 'reference' strain of biotype 2 (Bertschinger 2008/76), and two additional biotype 1 strains, strain BC181, which is less virulent than the type strain, and strain K17, which was isolated from a lamb, were investigated with respect to iron acquisition. All strains produced iron-repressible outer membrane proteins. However, only the type and biotype 2 strains could acquire iron from porcine transferrin and no organism could utilize human, bovine or ovine transferrin, or ovine or porcine lactoferrin; haemoglobin supported good growth of all strains except strain K17. In all cases, iron acquisition from transferrin and haemoglobin required direct contact between the organisms and the proteins indicating the existence of specific receptors. An affinity isolation technique, using biotinylated porcine transferrin plus streptavidin-agarose, allowed the isolation of the following polypeptides from total membranes of organisms grown under iron-restricted conditions: 99 kDa and 64 kDa from ATCC 27088; 93 kDa from Bertschinger 2008/76; 95 kDa (trace amounts) and 60 kDa from BC181; none from K17. These results indicate that the 93-99 kDa polypeptides are involved in the acquisition of iron from porcine transferrin and that the inability of strain K17 to use transferrin as an iron source is due, probably, to the lack of, or a defect in, an analogous component.

Actinobacillus pleuropneumoniae↗

Contact-dependent acquisition of transferrin-bound iron by two strains of Haemophilus parasuis.

Two strains of Haemophilus parasuis, namely, the type strain (ATCC 19417) and strain E751, were investigated with respect to iron acquisition. Both strains produced iron-repressible outer membrane proteins and could acquire iron from porcine transferrin but not from porcine lactoferrin. Neither strain used bovine transferrin, and human transferrin was used to only a very limited extent, if at all. In all cases, iron acquisition from transferrin required direct contact between the organisms and the protein. An affinity isolation technique based on biotinylated porcine transferrin plus streptavidin-agarose, followed by SDS-PAGE, allowed the isolation and identification of two potential porcine transferrin binding polypeptides (94 and 60 kDa) from total membranes derived from the type strain grown under iron-restricted conditions but only one (96 kDa) from strain E751. Each of these polypeptides was iron repressible and was not isolated when biotinylated human or bovine transferrin was used instead of biotinylated porcine transferrin. It is concluded that both strains acquire transferrin-bound iron by means of siderophore-independent mechanisms and that the isolated polypeptides represent porcine transferrin receptor components.

Animals↗

Effect of haemin limitation on the cytochrome complement and glucose metabolism of non-typable Haemophilus influenzae.

Haemophilus influenzae grown to exponential phase or stationary phase in medium with a low initial concentration of haemin (0.25 microgram/ml) was virtually devoid of cytochromes. Compared with bacteria grown in the presence of excess haemin (10 micrograms/ml), the haemin-limited organisms failed to respire formate and succinate and, generally, the respiratory rates with other substrates were reduced. However, growth rates were not affected by the haemin supply. Haemin-limited growth was associated with a reduced efficiency of glucose utilisation, in terms of glucose growth yields, and affected the net levels of excreted organic acids. Haemin limitation resulted in reduced acetate and increased succinate accumulation in the culture medium and the novel presence of D-lactate. These results indicate that, in contrast to the phenotype expressed in vitro during conventional cultivation of H. influenzae, the haemin-limited phenotype, which may be expressed in vivo, is characterised by a lack of cytochromes and a shift towards a more anaerobic type of metabolism.

Bronchiectasis↗

Effect of oxygen supply during growth on the production of cytochromes, enzymes, and acid end products by Haemophilus parasuis.

Haemophilus parasuis, grown under conditions of high aeration, was found to lack a tricarboxylic acid cycle but to possess phosphoenolpyruvate carboxylase and a reductive pathway leading to the production of succinate. Such organisms contained approximately equal quantities of b-, c-, and d-type cytochromes and excreted acetate. When the oxygen supply for growth was either reduced or eliminated, the specific activities of phosphoenolpyruvate carboxylase, malate dehydrogenase, fumarase, fumarate reductase, and NADH: fumarate oxidoreductase were increased substantially, and the acid products were succinate, acetate, and formate. Organisms grown under the latter conditions also contained increased quantities of b- and c-type cytochromes, some of which were low-potential cytochromes. These low-potential cytochromes were reduced by NADH and oxidized by fumarate, and hence, appeared to be components of NADH: furmarate oxidoreductase. Our results indicate that in H. parasuis, growing aerobically in medium containing glucose, the sole function of the reductive pathway is to provide intermediates for biosynthetic processes, and oxygen is the preferred electron acceptor. As the supply of oxygen is reduced or eliminated, the reductive pathway becomes more involved in NAD+ recycling and fumarate becomes the acceptor. In effect, irrespective of the oxygen supply, the growth of H. parasuis is absolutely dependent upon the presence of an electron transport system.

Acetates↗

Phenotypic variation in the outer membrane protein composition of Actinobacillus (Haemophilus) pleuropneumoniae: non-specific effect of exogenous pyridine nucleotide supply.

Actinobacillus pleuropneumoniae, grown in batch culture, was provided with pyridine nucleotides at concentrations that limited the final growth yield (pyridine nucleotide-deficient cultures) or did not determine the final extent of growth (pyridine nucleotide-sufficient cultures). Sarkosyl-extracted outer membranes from stationary phase, pyridine nucleotide-sufficient organisms contained 23,000 Mr and 43,000 Mr polypeptides that were absent (23,000 Mr) or barely detectable (43,000 Mr) in outer membranes from stationary phase, pyridine nucleotide-deficient organisms or exponential phase organisms. When growth ceased due to exhaustion of pyridine nucleotide, the ratio of the major outer membrane polypeptides (31,000, 38,000 and 69,000 Mr) was altered, becoming more like the ratio found with exponential phase organisms. Similar results were obtained when growth ceased due to glucose exhaustion at low biomass concentrations demonstrating that diverse nutrient deprivations can induce similar changes in outer membrane protein profile. All of these polypeptides were recognized by porcine immune sera indicating their production by A. pleuropneumoniae growing in vivo.

Actinobacillus pleuropneumoniae↗

Isolation and identification of a putative porcine transferrin receptor from Actinobacillus pleuropneumoniae biotype 1.

Each of two affinity isolation methods, the first based on biotinylated porcine transferrin plus streptavidin-agarose, and the second on Sepharose-coupled porcine transferrin, followed by SDS-PAGE, allowed the isolation and identification of two potential porcine-transferrin-binding polypeptides (approximately 64 kDa and 99 kDa) from total membranes of Actinobacillus pleuropneumoniae grown under iron-restricted conditions. Both polypeptides were iron-repressible and were identified as potential receptor candidates as they were not isolated when biotinylated human transferrin was used instead of biotinylated porcine transferrin. The 64 kDa polypeptide was the more easily removed from Sepharose-coupled porcine transferrin and only the 99 kDa polypeptide appeared to be an outer-membrane protein. While these results suggest that the 99 kDa polypeptide represents the porcine transferrin receptor of A. pleuropneumoniae, and that the 64 kDa polypeptide represents an associated protein serving an accessory role, other interpretations are also possible.

Actinobacillus pleuropneumoniae↗

Responses of Haemophilus pleuropneumoniae to iron restriction: changes in the outer membrane protein profile and the removal of iron from porcine transferrin.

Outer membranes from Haemophilus pleuropneumoniae grown under iron-replete and iron-restricted conditions in vitro were analysed by means of SDS-PAGE and immunoblotting. Iron restriction resulted in the appearance of two or more novel polypeptides in the molecular size range of 96-102 kD and an increased amount of a 79 kD polypeptide. These polypeptides were recognized by porcine immune sera indicating their production by H. pleuropneumoniae during growth in vivo. Although soluble siderophore production could not be detected, growth of the organisms on an iron-restricted medium was enhanced by the presence of porcine transferrin but not by bovine or human transferrin. The results suggest that H. pleuropneumoniae possesses a specific transferrin receptor, perhaps in the form of an iron-regulated outer membrane protein.

Animals↗

Defining the metabolic and growth responses of porcine haemophili to exogenous pyridine nucleotides and precursors.

A variety of biologically important pyridine nucleotides and precursors were examined for their capacities to satisfy the V-factor requirement of 30 strains of porcine haemophili. Of the compounds tested, only NAD, NMN and nicotinamide riboside (NR) supported the growth of all strains; NADP supported the growth of only the type strain of Haemophilus parasuis. Further studies with the H. parasuis type strain and the neotype strain of H. pleuropneumoniae demonstrated that, during growth, these organisms exhibited affinities for NMN that were greater than those for NAD; the affinity of H. pleuropneumoniae for NR was similar to that for NMN, whereas H. parasuis exhibited relatively low affinity for NR. With either organism, equimolar amounts of NAD and NMN supported the production of approximately equal amounts of biomass whereas growth yields were substantially lower when NR was the pyridine nucleotide source. When either organism was grown in the presence of excess exogenous [carbonyl-14C]NAD, cessation of growth was accompanied by the apparent exhaustion of the NAD supply. Approximately 80% of the radioactivity added as [14C]NAD could be recovered as extracellular [14C]nicotinamide and the majority of the assimilated radioactive material was present intracellularly in the form of a [14C]NAD(P) pool. The results are discussed in terms of the structural features required of a pyridine compound for it to support the growth of porcine haemophili, the capacity of these organisms to compete for pyridine nucleotide sources in vivo, and possible mechanisms involved in the assimilation of such compounds.

Animals↗

Pyridine nucleotide metabolism by extracts derived from Haemophilus parasuis and H. pleuropneumoniae.

A variety of biologically important pyridine nucleotides and precursors were examined for their capacities to serve as substrates for the synthesis of NAD by cell fractions derived from Haemophilus parasuis and H. pleuropneumoniae. Of the compounds tested, only NMN and nicotinamide riboside were converted to NAD. These reactions required ATP as co-substrate, and fractions from both organisms could also catalyze the ATP-dependent synthesis of NADP from NAD. In the absence of ATP, and depending on the pyridine compound under study, NAD, NMN, nicotinamide riboside, and also nicotinamide, were detected as products of catabolism. It is concluded that these haemophili possess either three-membered pyridine nucleotide cycles or two-membered cycles with synthetic branches originating with nicotinamide riboside. It is also possible that the pyridine nucleotide cycles of both organisms have nonrecycling branches resulting in the "waste" of usable pyridine compound in the form of nicotinamide.

Adenosine Triphosphate↗

Tryptone-yeast extract broth as a culture medium for Haemophilus pleuropneumoniae and Haemophilus parasuis to be used as challenge inocula.

Haemophilus pleuropneumoniae and Haemophilus parasuis were grown in a liquid medium containing tryptone and yeast extract. Whereas culture turbidity during exponential growth of either organism was proportional to the number of viable cells, continued incubation of stationary phase cultures was accompanied by a dramatic decrease in viability with no corresponding decrease in culture turbidity. It is concluded that the medium described should prove useful for the growth of porcine haemophili for use in experimental infections provided the stage of growth is given due consideration.

Animals↗