Ascaris suum: Oxidative phosphorylation in mitochondria from developing eggs and adult muscle

Respiration and the phosphorylating capability of mitochondria isolated from one-celled fertilized eggs, 10-day vermiform embryos, 21-day infective larvae, and adult body wall muscle from Ascaris suum were compared with that of rat liver mitochondria. Although oligomycin-sensitive ATPase and O₂ consumption/ mitochondrion in the presence of succinate and malate was lower in eggs than in liver, other properties such as respiratory control, ADP:O and P:O ratios at sites I, II, III, and the sensitivity of respiration to cyanide, azide, oligomycin, rotenone, and malonate were similar. In muscle mitochondria, the oligomycin-sensitive ATPase and O₂ consumption/ mitochondrion were sharply reduced, respiratory control was poor, and electron transport at sites II and III in particular was inefficiently coupled with phosphorylation. In addition, about 60% of the respiration was insensitive to cyanide or azide but sensitive to salicylhydroxamic acid. The results support earlier evidence that the free-living eggs of A. suum are aerobes. The adult parasite, while continuing to ferment actively in the presence of oxygen, nevertheless possesses one or more electron transport systems that are inefficiently coupled with aerobic phosphyorylations. The physiological significance of these systems has yet to be elucidated.     

Ascaris suum: Free amino acids and proteins in the pseudocoelom,seminal vesicle and glandular vas deferens

The free amino acids and proteins of the seminal vesicle and pseudocoelomic fluids in the male Ascaris suum were examined and compared. The seminal fluid contained a high concentration of lysine (lysine: glutamic acid ratio of 5:1) while the pseudocoelomic fluid contained more glutamic acid than lysine with alanine, serine, glycine and proline being the most abundant free amino acids. The proteins present in the seminal fluid differed from those in the pseudocoelomic fluid in both number and molecular weight. The sperm activating substance (SAS) present in homogenates of the glandular vas deferens of male worms is nondialyzable, heat-sensitive and can be precipitated using 45% saturated ammonium sulfate. Active moieties can be recovered following passage of the ammonium sulfate precipitates through ultrafiltration membranes or by applying gland homogenates to an ion exchange column. When subjected to SDS-polyacrylamide gel electrophoresis, the active fractions revealed both low and high molecular weight substances. During attempts to purify a single activating substance, it was noted that the more heterogeneous fractions contained the highest activating capacity. Thus, no precise relationship between the biological activity and the purity of the various fractions was determined.     

Ascaris suum: Nutrient absorption,growth, and intestinal pathology in young pigs experimentally infected with 15-day-old larvae

Three-week-old pigs on high (HP) or low (LP) protein diets were infected with 15-day-old Ascaris suum larvae (W). Including noninfected pigs (C), the experimental groups were HPW, LPW, HPC, and LPC. After 8 weeks, worm burden in the intestine averaged 42 in LPW and 31 in HPW. Nitrogen balance during Week 4 showed nonsignificantly less nitrogen absorption and retention in LPW compared to LPC. A similar, nonsignificant decrease in fat absorption was recorded in LPW vs LPC and in HPW vs HPC. The weight of the small intestine was significantly greater in W than C pigs but did not differ because of protein level. The weight correlated positively to worm burden and the increase was due mainly to hypertrophy of the tunica muscularis (muscle layers).     

Nippostrongylus brasiliensis: Effect of cyclophosphamide treatment on worm expulsion in rats

The precise immunological mechanisms associated with expulsion of the gastrointestinal nematode Nippostrongylus brasiliensis remain controversial. In order to investigate the effects of drug-induced immunosuppression on parasite burdens and expulsion, various regimens of cyclophosphamide were administered to parasitized Wistar rats. It was observed that both the number of worms established from an infective dose of 3000 larvae and the time of expulsion were markedly increased with higher doses of cyclophosphamide. Thus, at the highest sublethal level of treatment (100 mg/kg), 82% of the infective dose was recovered at Day 9 postinfection compared with 51% in nontreated controls. Furthermore, in such treated rats expulsion was delayed in 6 days beyond that of nontreated animals. As cyclophosphamide, at the levels used in the present study, is known to primarily effect B-cell function, the results support the view that antibody-mediated responses play an essential role in worm expulsion.     

Avermectin B1a: an irreversible activator of the gamma-aminobutyric acid-benzodiazepine-chloride-ionophore receptor complex

Avermectin B_1a, an antihelminthic macrocyclic lactone, has been previously shown to reduce muscle membrane resistance by stimulating γ-aminobutyric acid-mediated chloride conductance. Since the benzodiazepine receptor is coupled to a receptor for γ-aminobutyric acid and related chloride ionophore, the effects of Avermectin B_1a on [³H]diazepam binding to the benzodiazepine receptor were studied. In well-washed membrane fragments from rat cerebral cortex, Avermectin B_1a markedly increased the binding of [³H]diazepam to benzodiazepine receptors. This effect was qualitatively similar to that observed with either γ-aminobutyric acid or chloride ion and was partially reversed by the γ-aminobutyric acid receptor antagonist, bicuculline. In contrast to the effects of γ-aminobutyric acid and chloride, the enhanced binding of [³H]benzodiazepine elicited by Avermectin B_1a was $\text{not}$ reversed by extensive washing of the membrane preparation. Avermectin B_1a appears to irreversibly modify benzodiazepine receptors at a γ-aminobutyric acid-chloride recognition site and may be valuable in biochemical studies of the regulation of benzodiazepine receptor function.     

Eimeria nieschulzi and Trichinella spiralis: Analysis of concurrent infection in the rat

The effects of concurrent primary infection of the rat with Eimeria nieschulzi and Trichinella spiralis on the number of oocysts of E. nieschulzi shed by the host and on the number, distribution, and fecundity of adult T. spiralis were analyzed. When rats were initially infected with E. nieschulzi followed 9 days later by infection with T. spiralis there occurred a significant decrease in the total numbers of adult worms in the small intestine, a significant shift in the position of these worms along the length of the small gut, a decrease in the fecundity of adult female worms, and a decrease in muscle parasitism when compared with rats infected with T. spiralis alone. When rats were initially infected with T. spiralis, followed 9 days later by infection with E. nieschulzi, there occurred a significant decrease in the numbers of oocysts shed over 24 hr on Days 7, 9, and 11 postinfection below that seen with rats infected only with Eimeria. These changes are discussed in terms of the enteropathophysiologic lesions and enteric inflammation known to occur during infections with these two parasites.     

Angiostrongylus cantonensis: Biogenic amines in the lungs of infected rats

Levels of histamine, serotonin, norepinephrine, and dopamine were estimated sequentially in rats parasitized by the lungworm, Angiostrongylus cantonensis, between 30 and 75 days postinfection. The highest level of histamine in the infected lungs was 52.19 μg/g wet wt tissue, 13 times higher than the level found in control rats. The level of serotonin rose from the normal level of 6.41 to 10.27 μg/g wet wt tissue after the worms had lodged in the pulmonary artery for 15 days. There were no changes in norephinephrine or dopamine. Studies of host cell response to infection revealed that the increased histamine and serotonin levels corresponded to a rise in the lung population of mast cells, suggesting that these cells produced the amines.     

Litomosoides carinii: Effect of splenectomy on the ability of naive Mastomys natalensis to accept transplanted adults

Fifty-eight male rats Mastomys natalensis, were used in each of two experiments to study the effect of splenectomy on the rejection of transplnted nematodes, Litomosoides carinii. The rats were divided into five groups: splenectomized recipients (SR), normal recipients (NR), splenectomized (S), normal (N) and donor (D). Each group had 12 animals except for the D group which had 10. After patency of the D group (tank infected with Litomosoides carinii), groups SR and S were splenectomized. Fourteen days later, (three to five L. Carinii derived from the D group) were surgically transplanted into the peritoneal cavity of the SR and NR groups. In the first experiment, weekly sacrifices were made starting at Day 3. In the second experiment, all groups were sacrificed at Day 32. Worms transplanted into the SR group were accepted while those transplanted into the NR group were rejected. Sequentially examined antibody titers after Day 3 fell into two groups, those that were recipients of transplants (SR and NR) and those that were not (NR and SR). After becoming positive on Day 3, the microfilaremia of the SR group rose by Day 31 while that of the NR group fell to near 0. It was concluded that the spleen is necessary for the rejection of transplantedL. carinii by naive M. natalensis.     

Trichinella spiralis: Genetics of worm expulsion in inbred and F1 mice infected with different worm doses

The nematode Trichinella spiralis is rejected from the intestine at a time that is characteristic for each inbred strain of mouse. Previous work (R. G. Bell et al. 1982a) had empirically identified strong, intermediate, and weak phenotypes (NFR, $\text{C}\text{H}\text{He}$, and $\text{C57}\text{10}$ mice, respectively) in mice infected with 400 muscle larvae. It is shown that this classification applies to another eight inbred strains: SWR, $\text{DBA}\text{2}$, $\text{DBA}\text{1}$, LP, $\text{Bub}\text{Bn}$—all intermediate, and $\text{NZB}\text{BIN}$, C57L, A, and Mus molossinus—all weak. This phenotypic classification consistently applies with infections of 400–800 muscle larvae. Below doses of 300 muscle larvae, the strain designation of phenotype does not consistently apply. By this it is meant that the relative rejection rate changes for certain strains so that eventually some strains that were strong (NFR) or intermediate (AKR) responders to 400 muscle larvae become weak responders to 50 muscle larvae. Other strains increase their relative rejection time (B10 · BR, B10 · Q) while many do not change (NFS, $\text{C}\text{3}\text{Heb}\text{Fe}$, $\text{DBA}\text{2}$, $\text{DBA}\text{1}$). The phenomenon is most apparent in inbred parental strains rather than in F₁ crosses, and it represents a phenotypic variation in rejection time that is dependent on dose. It is also demonstrated that time of rejection is directly proportional to dose in all inbred and F₁ mouse strains that we have examined. Analysis of F₁ crosses shows that most have the rejection time of the strongest responding parental line, suggesting simple genetic control of strong, intermediate, and weak responses. Two F₁ crosses invalidated this theory. The $\text{DBA}\text{1}\text{×}\text{C3}\text{H}\text{He}$ (intermediate × intermediate) showed a strong response. The additive effects of parental rejection phenotype indicated that these lines could not be genetically identical for intermediate responsiveness. Similarly, the NFR (strong) × B10 · BR (weak) F₁ showed intermediate rejection, indicating partial dominance of $\text{C}\text{57}\text{B}\text{1}\text{10}$ genes over the strong responder NFR strain. Neither the primary expulsion time phenotype, phenotypic variation to low doses, or the rejection characteristics of F₁ crosses could be ascribed to genes linked to the major histocompatibility complex.     

Neoaplectana carpocapsae: Movements of nematode populations on a thermal gradient

To gain information on factors which could affect the nematode Neoaplectana carpocapsae's, dispersion and infection of insect larvae in the field, nematode populations were tested on a thermal gradient (0.5 C/cm). Infective juveniles grown at 15, 20, and 25 C migrated on the gradient toward their respective growth temperatures when tested immediately after harvesting from their medium. This migration by juveniles, grown at 20 or 25 C, was altered within 12 hr by shifting incubation temperature. The nematodes' tendency to migrate toward warmer temperatures, when placed below incubation temperature, decreased during 6–7 days incubation at 20 or 25 C and the nematodes reversed direction: incubation at 2–5 C inhibited this reversal. Nematodes incubated at 20 C, then applied to a gradient zone at 12–13 C, had a greater tendency (P < 0.05) to remain aggregated in that zone within a 3-hr period than those applied at 17–18, 22–23, or 27–28 C. Their tendency to migrate from the 12–13 C zone was significantly (P < 0.05) increased by shifting them to 15 C for 14–138 hr.     

Strongyloides ratti: Dissociation of the rat's protective immunity into systemic and intestinal components

Analysis of the early stages of a challenge infection with Strongyloides ratti has shown that protection is expressed against the developing third-stage larval worms (L₃) and prevents the maturation to adulthood of most larvae. Challenge after an immunizing infection that was restricted to the parenteral L₃ migratory phase showed that some 10–40% of overall protection could be ascribed to systemic antilarval immunity. Some larvae were trapped in the skin at the site of injection whereas others failed to migrate to the head and lung of immune rats. Larvae arriving in the intestine at Days 3, 4, and 5 did not persist beyond Day 7 and 8. Studies using [⁷⁵Se]methionine-labeled L₃ showed a significant increase in fecal label in rats immunized by a complete infection. This loss did not occur to the same extent in rats immunized only with parenteral larvae. Significant rejection of worms transplanted to the intestine also indicated intestinal protection. The possible existence of large numbers of worms in a state of “arrested development” was excluded by their failure to appear after cortisone treatment and the absence of worm accumulation in radiolabeling studies. It is concluded that at least two responses operate against larval S. ratti, one is systemic and the other operates in the intestine against larvae in a manner that resembles the “rapid expulsion” rejection of Trichinella spiralis in immune rats.     

Elaphostrongylus rangiferi: Influence of temperature,substrate, and larval age on the infection rate in the intermediate snail host, Aarianta arbustorum

The infection rate of the first stage larval nematodes, Elaphostrongylus rangiferi, was studied experimentally, using the juvenile snail Arianta arbustorum as intermediate host. The nematode showed a linear, fivefold increase in infection rate within the temperature range of 4 to 28 C. The snails were exposed to the larval nematodes on three different substrates. The highest infection rate was recorded when snails were exposed in tap water and significantly slower infection rates were obtained when either lettuce or soil was used as the substrate. First stage larvae of E. rangiferi were infective for at least 2 months when stored at 12 C. Throughout this period, the infection rate showed a significant decline, while the motility of the larvae remained unchanged.     

Strongyloides ratti: Structural and functional characteristics of normal and immune-damaged worms

Adult Strongyloides ratti recovered at Day 6 of a primary infection in the rat appear normal in terms of ultrastructural morphology; the occurrence of membranous material within the gut lumen of the nematode indicates that such specimens are feeding. As the infection progresses, degenerate changes occur in the worm tissues. Lipid droplets and dense granules accumulate in intestinal cells. The contents of the gut lumen indicate that worms continue to feed until at least Day 20, but thereafter, as they migrate from the anterior to the posterior half of the intestine, feeding ceases. This is associated with the development of oral plugs, which contain host immunoglobulins, and may represent antigen-antibody complexes. Damaged worms are considerably smaller than 6-day normal worms, but this stunting is not reflected by a change in the thickness of the cuticle. Worms recovered 7 days after a challenge infection do not differ significantly from specimens recovered at the end of a primary infection. A seemingly unique feature of S. ratti is the existence of a “margination membrane” which delimits the brush border glycocalyx. This membrane shows unilaminar or multilaminar configurations, unlike a true lipid bilayer; possibly it is secreted by the parasite.     

Trichinella spiralis: Characterization and strain distribution of rapid expulsion in inbred mice

Appropriately immunized mice display a response that is biologically equivalent to rat rapid expulsion. Only two inbred strains ($\text{NFR}\text{N}\text{and}\text{NFS}\text{N}$ derived from NIH Swiss mice) have been shown to respond in this manner. Mice of the $\text{Balb}\text{c}$, CBA, $\text{A}\text{He}$, C₃H, SJL, or C₅₇Bl strains are “nonresponders” which require approximately twice as much intestinal exposure (in days) to Trichinella spiralis to elicit a response half as effective. Genetically, the responder is dominant, autosomal, and does not appear to be linked to the MHC. The characteristics of mouse and rat rapid expulsion of T. spiralis are not identical but share these features: initial rejection within 24 hr of challenge; a rejection efficiency >90%, from 1 to 5 weeks after the primary; induction of response does not require exposure to the complete infection; rapid expulsion is immunologically specific for preadults; adult worms are resistant. While a genetic basis for responsiveness exists in mice there is, as yet, no evidence for genetic control in rats. In both mice and rats, rapid expulsion is distinguished from the intestinal hyperreactivity associated with rejection of the primary infection by the kinetics and amplitude of the rejection of transplanted adult worms.     

Dipetalonema viteae: Response of spleen cells in experimental mouse filariasis to mitogens and antigens

Balb/c mice were infected by transplanting 3, 5, 10, or 20 female adult Dipetalonema viteae under the dorsal skin. The microfilaremias resulting from infections with 3 or 5 adult worms were of lesser magnitude and of shorter duration than those produced in infections with 10 or 20 worms. Spleen cells taken from these mice at various intervals after infection were assayed in vitro for their ability to respond to phytohemagglutinin (PHA) or lipopolysaccharide (LPS). There was no depression in the response to LPS or to PHA in mice given infections of 3 or 5 D. viteae adult worms. In contrast, the response to PHA was significantly depressed in groups receiving 10 or 20 adult female worms 12 days after infection and by Day 25, the depression was severe. Thereafter the PHA responsiveness recovered gradually to reach control values on Day 60. In mice transplanted with 10 or 20 D. viteae adult worms there was no significant depression in the response to LPS at any time during the infection, but the response was increased slightly sporadically during infection. These results indicate that in mice, this infection causes an initial suppression in the function of PHA-sensitive T cells but has little effect on the B cells which respond to LPS. A factor present in serum taken on Day 25 from mice infected with 10 or 20 adult worms inhibited the proliferative response to PHA by spleen cells from normal mice. The recovery of PHA responsiveness in mice given the heavier infections coincided with death of the adult worms, but mitogen reactivity and microfilaremia were unrelated. Antigens from male or female worms induced cell division in spleen cells taken from infected mice after microfilaremia had ceased whether they were implanted with 3 or 10 adult worms.     

Caenorhabditis elegans: Characters of negatively charged groups on the cuticle and intestine

Partial characterization of carboxyl, sulfate, and phosphate groups on the Caenorhabditis elegans cuticle and intestinal microvilli was achieved by en face labeling of floating cryosections at two pH levels and specific blockage of sulfate groups by Alcian blue. All negatively charged groups on the cuticle and intestinal microvilli labeled heavily at pH 7.2–7.4. Pretreatment to block sulfate groups followed by ferritin labeling at pH 7.2–7.4 gave a 35% reduction of binding on the cuticle and an 80% reduction in binding on the microvilli. At pH 1.8 or 2.5, only the sulfate groups labeled as shown by the complete abolition of labeling on the cuticle and the microvilli following blockage of the sulfate groups. Molecules with accessible sulfate groups were distributed in clusters throughout the cortical layer of the cuticle, were present in the struts of the median layer but were absent from the basal layer. The advantages of applying molecular probes to cryosections as compared to sections prepared by standard electron microscopical techniques are discussed.     

Trypanosoma brucei: Nearest neighbor analysis on the major variable surface coat glycoprotein—Crosslinking patterns with intact cells

Cleavable Crosslinking reagents were used to study interactions among proteins of the surface coat of Trypanosoma brucei. The proteins were resolved by two-dimensional polyacrylamide gel electrophoresis in sodium dodecyl sulfate. When intact cells were treated with dithiobis(succinimidylpropionate), we obtained extensive intermolecular Crosslinking of major variable surface coat glycoprotein (VSCG) molecules. This reagent generated no apparent crosslinks between VSCG and other membrane-associated proteins. Complete conversion to oligomers equal to or greater than octamers occurred within 20 min. When purified VSCG in solution was treated with dithiobis(succimidylpropionate), dimers were found. A complex of Cu²⁺ and 1,10-phenanthroline was used to catalyze air oxidation of adjacent sulfhydryls to disulfide bonds; however, no crosslinking among VSCG molecules nor between VSCG and other proteins was observed. The results presented indicate that VSCG in solution exists predominately in the form of dimers. Whether VSCG in situ also occurred as dimers could not be determined; however, since we observed trimeric and tetrameric forms of VSCG when untreated cells were analyzed, it is likely that weak interactions occur among the protein molecules. These interactions are less stable than the dimer association observed with purified VSCG. Finally, the analysis indicated that VSCGs of this stock of T. brucei, derived from UGANDA/ 60/TREU/164[ETat3], contained at least one intramolecular disulfide bond. We examined T. brucei stocks 427 and EATRO 110 and obtained similar results. Thus, it appears that intramolecular disulfide bonding is a general feature of T. brucei VSCGs.     

Virus-induced gene silencing of PEAM4 affects floral morphology by altering the expression pattern of PsSOC1a and PsPVP in pea

pea-MADS4 (PEAM4) regulates floral morphology in Pisum sativum L., however, its molecular mechanisms still remain unclear. Virus-induced gene silencing (VIGS) is a recently developed reverse genetic approach that facilities an easier and more rapid study of gene functions. In this study, the PEAM4 gene was effectively silenced by VIGS using a pea early browning virus (PEBV) in wild type pea JI992. The infected plants showed abnormal phenotypes, as the floral organs, especially the sepals and petals changed in both size and shape, which made the corolla less closed. The petals changed in morphology and internal symmetry with, the stamens reduced and carpel dehisced. Larger sepals and longer tendrils with small cauline leaves appeared, with some sepals turning into bracts, and secondary inflorescences with fused floral organs were formed, indicating a flower-to-inflorescence change. The infected plants also displayed a delayed and prolonged flowering time. The PEAM4-VIGS plants with altered floral morphology were similar to the pim (proliferating inflorescence meristem) mutant and also mimicked the phenotypes of ap1 mutants in Arabidopsis. The expression pattern of the homologous genes PsSOC1a and PsSVP, which were involved in flowering time and florescence morphological control downstream of PEAM4, were analyzed by real-time RT-PCR and mRNA in situ hybridization. PsSOC1a and PsSVP were ectopically expressed and enhanced in the floral meristems from PEAM4-silenced plants. Our data suggests that PEAM4 may have a similar molecular mechanism as AtAP1, which inhibits the expression of PsSOC1a and PsSVP in the floral meristem from the early stages of flower development. As such, in this way PEAM4 plays a crucial role in maintaining floral organ identity and flower development in pea.     

Association of the eukaryotic V1VO ATPase subunits a with d and d with A

Owing to the complex nature of V₁V_O ATPases, identification of neighboring subunits is essential for mechanistic understanding of this enzyme. Here, we describe the links between the V₁ headpiece and the V_O-domain of the yeast V₁V_O ATPase via subunit A and d as well as the V_O subunits a and d using surface plasmon resonance and fluorescence correlation spectroscopy. Binding constants of about 60 and 200 nM have been determined for the a-d and d-A assembly, respectively. The data are discussed in light of subunit a and d forming a peripheral stalk, connecting the catalytic A₃B₃ hexamer with V_O.

Structured summary

MINT-7012054: d (uniprotkb:P32366) binds (MI:0407) to A (uniprotkb:P17255) by fluorescence correlation spectroscopy (MI:0052)MINT-7012041: d (uniprotkb:P32366) binds (MI:0407) to A (uniprotkb:P17255) by surface plasmon resonance (MI:0107)MINT-7012028: d (uniprotkb:P32366) binds (MI:0407) to a (uniprotkb:P32563) by surface plasmon resonance (MI:0107)     

Plasmodium yoelii: Antibody and the maintenance of immunity in BALB/c mice

Subpatent persistence of parasitemia was detected for up to 7 weeks after infection of BALB/c mice with Plasmodium yoelii. Serum taken from recovered mice maintained parasitemias in recipient mice at a subpatent level when transferred repeatedly at 2-day intervals. Single doses of serum from convalescent donors delayed the course of infection in recipients. Small doses of transferred hyperimmune serum had the same effect, whereas large doses (>0.5 ml) totally suppressed parasitemia. Only a single secondary challenge of recovered mice was required in order to produce a maximally protective hyperimmune serum. Mice completely protected from a primary challenge with P. yoelii by transfer of hyperimmune serum were not at all resistant to a second challenge given some weeks later. After transfer of hyperimmune serum into mice with established P. yoelii infection, parasitemia fell to subpatent levels within 48 hr. During the first 21 hr after serum transfer, a progressive reduction in the proportion of ring forms present in blood smears was observed.