Low molecular weight immunosuppressors secreted by adult Nematospiroides dubius

Adult Nematospiroides dubius excretory-secretory products (ES) were collected from worms cultured in vitro, separated by sodium dodecyl sulphate--polyacrylamide gel electrophoresis (SDS-PAGE) into four fractions (FI-IV), electroeluted and assessed for their ability to inhibit the proliferation of mouse lymphocytes stimulated by mitogens in vitro. The proliferation of mitogen- and ES-stimulated mouse spleen lymphocytes from normal and infected mice was inhibited by low mol. wt ES F-IV (less than 26,000).     

Proteolytic enzymes in excretory-secretory products from adult Nematospiroides dubius

Excretory-secretory products (ES), collected from in vitro cultures of adult Nematospiroides dubius, were examined for proteolytic enzyme activity. ES enzymes had a pH optimum of 8.0 and their activity was sensitive to serine-proteinase inhibitors. Three SDS-resistant proteases were identified in ES at molecular weight (mol. wt) 200,000, 105,000 and 48,000 by incorporating substrates into the matrices of sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE) gels.     

The use of electroblotted antigens of Trichostrongylus colubriformis to induce proliferative responses in sensitized lymphocytes from sheep

Merino sheep were immunized against the intestinal nematode, T. colubriformis, by repeated infections, and proliferative responses of their peripheral blood lymphocytes (PBL) against parasite extracts and excretory-secretory (ES) antigens were monitored over 130 days. Maximal responses occurred 7-14 days after challenge. The ability of soluble proteins and parasite antigens to induce proliferation was compared with that of antigen-bearing particles obtained after antigen was adsorbed onto nitrocellulose. Blank particles increased c.p.m. two- to three-fold above that obtained in medium alone, and to elicit proliferative responses of comparable magnitude between 10 and 100 times more antigen was required when antigen-bearing particles were used instead of soluble extracts or defined proteins. Blood leucocytes as well as T-cell lines established by stimulation with parasite antigens in vitro reacted to moieties of from 5000 to 38,000 mol. wt in ES antigens on nitrocellulose particles. Direct comparisons of T-lymphocyte responses with antibody responses as assessed by immunoblots revealed different profiles of immunogenicity among ES proteins within individual sheep, but the 10,000, 30,000 and 75,000-90,000 mol. wt proteins were immunodominant. These proteins were also those consistently recognized by T-lymphocytes and sera from sheep immunized with ES proteins in adjuvant. Thus, this technique can be applied to identify parasite material which is immunogenic for T-lymphocytes, but the sensitivity of the procedure in sheep is less than reported in human studies.     

Characterization of molecules involved in protein translocation using a specific antibody

The vectorial translocation of nascent proteins through the membrane of the rough endoplasmic reticulum has been shown to require a specific membrane-bound protein whose cytoplasmic domain can be proteolytically cleaved and isolated as an active peptide of mol wt 60,000 (Meyer and Dobberstein, 1980, J. Cell Biol. 87:503-508). Rabbit antibodies raised against this peptide were used to further characterize the membrane- bound molecule. Immunoprecipitation of solubilized, radiolabeled rough microsomal proteins yielded a single polypeptide of mol wt 72,000, representing the membrane-bound protein from which the 60,000-mol wt peptide was proteolytically derived. The antibody could also be used to remove exclusively the 60,000-mol wt peptide, and thus the translocation activity, from elastase digests tested in a reconstituted system. Moreover, immunoprecipitation of elastase extracts alkylated with [14C] N-ethylmaleimide selected a single species of mol wt 60,000. Immunoprecipitation of in vivo radiolabeled proteins from the appropriate cell type yielded the 72,000-mol wt membrane protein irrespective of the duration of labeling, or if followed by a chase. Subsequent treatment with protease generated the 60,000-mol wt fragment. In addition, the antibody could be used to visualize reticular structures in intact cells which correspond to endoplasmic reticulum at the ultrastructural level. It is thus clear that one membrane component required in the vectorial translocation of nascent secretory (and membrane) proteins is a peptide of mol wt 72,000.     

Nematospiroides dubius: direct and correlated responses to selection for high and low immune responsiveness in mice

High and low immune responder lines of mice were bred selectively from an allogeneic stock over 10 generations, based on their fecal parasite egg count assayed 3 weeks after reinfection with 100 Nematospiroides dubius larvae. By generation 10, (F10), the low immune response mice voided about 10 times as many fecal N. dubius eggs as the high immune response mice. Realized heritability for the selected trait, fecal egg count after secondary infection (= protective immunity), was 0.35 at F7. F7 was considered the selection limit. Selection for change in fecal egg count did not significantly influence the conformational nor reproductive characteristics of these mice. Significant phenotypic and genetic correlations were evident between the selected character and innate immunity to N. dubius, humoral antibody response to N. dubius infection, and establishment, growth, and reproduction of N. dubius in the selected mice.     

Nematospiroides dubius and Nippostrongylus brasiliensis: delayed type hypersensitivity responses to ovalbumin in the infected mouse

Mice (C57BL) infected with the intestinal nematode Nematospiroides dubius showed depressed delayed type hypersensitivity responses to ovalbumin administered subcutaneously in Freund's complete adjuvant. IgG and IgM responses to this inoculum were unaffected. It is unlikely that the depression arose from impairment of the ear test response because responses to an extract of the adult parasite were measurable and ear testing with lipopolysaccharide yielded normal responses in infected mice. Furthermore, mice immunized on the day of infection responded normally, whilst long term infected mice ear challenged with antigen pulsed macrophages gave depressed responses. The in vitro proliferative responses of cells from the spleens and from the lymph nodes draining the site of immunization were enhanced marginally by N. dubius infection. Furthermore, these cells induced normal or elevated adoptive delayed-type hypersensitivity and IgG responses in irradiated recipients. These findings suggest that N. dubius does not compromise the development of ovalbumin specific T cells involved in a delayed type hypersensitivity response. Evidence for the induction of suppressor cells by N. dubius is discussed, and the findings are compared with results obtained with Nippostrongylus brasiliensis, a parasite which is rejected rapidly from the mouse.     

Nematospiroides dubius: influence of adjuvants on immunity in mice vaccinated with antigens isolated by affinity chromatography from adult worms

Five adjuvants were examined for their ability to potentiate the immune response of mice to soluble antigens from adult Nematospiroides dubius prepared by affinity chromatography against antibodies from repeatedly infected mice as ligands (IMIgAg). Immunized mice were better protected against N. dubius by IMIgAg injected intraperitoneally with either pertussigen (75%) or aluminium hydroxide (Alum) (67%) as adjuvants than with Freud's complete (54%) or incomplete adjuvants (31%). Protection was correlated with elevated specific antibody values and with cellular responses. Quil A was toxic to recipient mice at the concentration used. Alum may be a more practical adjuvant than pertussigen, which may activate protective immunity only in specific recipient genotypes and oil-based adjuvants which appear to be less efficient, to vaccinate mice with soluble parasite antigens.     

Plant lectins detect age and region specific differences in cell surface carbohydrates and cell reassociation behavior of embryonic mouse cerebellar cells

When plated at high cell density in a microwell culture system, freshly dissociated embryonic mouse cerebellar cells assemble into reproducible, 3-dimensional patterns. The addition of the dimeric lectin Succinyl Concanavalin A blocks reversibly the formation of the microwell pattern, suggesting that cell surface carbohydrates affect the reassociation behavior of embryonic mouse cerebellar cells. Agglutination studes of dissociated cell populations harvested from different regions of the embryonic brain reveal that different lectins agglutinate cell populations from different embryonic brain regions. Cells from E13 cerebellum are agglutinated with Concanavalin A, wheat germ agglutinin, Ricinus communis agglutinin, mol wt 60,000, Ricinus communis agglutinin, mol wt 120,000, and Lens culinaris, but not by soybean agglutinin or a fucose-binding protein. Cells from the midbrain are agglutinated only with Concanavalin A, Ricinus communis agglutinin, mol wt 60,000 and Ricinus communis agglutinin, mol wt 120,000; those from the cerebral cortex are agglutinated only with Lens culinaris; and those from the medulla are agglutinated only with Ricinus communis agglutinin, mol wt 60,000, and Ricinus communis agglutinin, mol wt 120,000. In addition, agglutination of cerebellar cells with Concanavalin A, wheat germ agglutinin, and Ricinus communis agglutinin is diminished over the course of development from embryonic day 13 to postnatal day 7. These studies suggest regional differences in the cell surfaces of the developling brain that are further modulated during the differentiation of the tissues. On a poly(D-lysine) treated substrate in microwell cultures, cell migration is unique to the cerebellum of the 4 brain regions studied. Surfaces treated with carbohydrate-derivatized poly(D-lysine) are currently being tested for their efficacy as substrates for differential cell migration.     

Nematospiroides dubius: genetic control of immunity to infections of mice

Inbred strains of mice differ in their susceptibility and resistance to challenge infections with Nematospiroides dubius. In our studies, F1 hybrid mice from resistant SJL and susceptible CBA parents were resistant to N. dubius challenge infections. Only 22% of backcrosses to SJL were susceptible while backcrosses to CBA had a wide range of susceptibility. Male mice were more susceptible than female mice. In another experiment, inbred strains of mice were compared in their ability to resist N. dubius challenge infection: SJL and A.SW (H-2s) mice became resistant after one immunizing infection, A, A/He (both H-2a), as well as BALB/c and DBA/2 (both H-2d) mice became resistant after two immunizing infections, while C57BL/6 (H-2b), C3H/He, CBA, and AKR (H-2k) mice remained susceptible. The resistance to reinfections was characterized by reduction of worm burdens between Days 6 and 14 postinfection. It was concluded that (1) resistance to N. dubius challenge infections is inherited in a dominant fashion and that multiple genes may influence such response, which in turn might be modulated by the Y chromosome; (2) both MHC and non-MHC genes may influence, in conjunction with the number of exposures to parasite antigens, the resistance to challenge infections.     

Nematospiroides dubius: passive transfer of protective immunity to mice with monoclonal antibodies

Nine hybridoma cell lines secreting monoclonal antibodies specific for Nematospiroides dubius were produced by fusion of the mouse myeloma cell line NS-1 to either spleen cells or mesenteric lymph node cells from mice repeatedly infected with N. dubius. Seven of the antibodies were identified as IgM and two as IgG1. Each monoclonal antibody bound to polypeptide epitopes on both infective larvae (L3) and adult worms. However, five antibodies bound preferentially to L3 and three to adult worms. All nine antibodies reacted with high molecular weight protein antigens. Passive protective immunity in Balb/c mice was demonstrated with monoclonal antibodies Nd2 and Nd3 in ascites fluid which stunted both male and female worms and reduced parasite fecundity.     

Nematospiroides dubius: response of the late fourth-stage larva to anthelmintics in vitro

Approximately 60% of fourth-stage larvae of Nematospiroides dubius recovered from mice 6 days after infection, developed to the young adult stage when cultured over a 7-day period in a complex medium in vitro. Larvae at the late fourth stage of development were found to be highly susceptible to most broad spectrum anthelmintics under in vitro conditions, the benzimidazole, imidazothiazole, pyrimidine, isothiocyanate and macrocyclic lactone compounds all being active at very low concentrations. Narrow spectrum anthelmintics active only against ascarids, pinworms, filariae, cestodes or trematodes had little or no effect on these larvae. Ineffective also were those chlorinated hydrocarbon, substituted phenol and salicylanilide compounds known to affect Haemonchus but not trichostrongylid worms in general. It is concluded that in vitro assays employing larvae of N. dubius are useful for the stringent screening of compounds for broad spectrum antitrichostrongyle activity. Used in conjunction with in vivo screens employing N. dubius in mice, they also afford means for detecting intrinsic activity against the parasite in a system free from any complicating host pharmacokinetics.     

Structural characterization of the 9-10S estradiol receptor from calf uterus

An affinity chromatography-based method has been developed for estrogen receptor isolation which requires the inclusion of sodium molybdate in purification buffers for maintaining the large 9-10S form of the receptor. The protein products obtained from affinity chromatography of calf uterine receptor extracts or from extracts presaturated with estradiol have been analyzed by gel electrophoresis under denaturing conditions. Major estrogen sensitive proteins were peptides with Mr approximately 90,000, 65,000 and 50,000. Two additional proteins (60,000 and 53,000) of lower abundance and with demonstrated estrogen sensitivity were also observed. Affinity labeling with [3H]tamoxifen aziridine identified the Mr 65,000 protein as the estrogen receptor and suggested that the Mr 60,000, 53,000 and 50,000 peptide components were derived proteolytically from this parent unit. The 90,000 mol. wt component was readily dissociated from heparin-sepharose immobilized estrogen receptor by elution with low salt buffers without molybdate. Peptide mapping experiments indicated that the 90,000 mol. wt component was not related to the Mr 65,000 and 50,000 estrogen receptors, but confirmed the smaller binding unit to be a proteolytic fragment of the 65,000 mol. wt receptor. The results suggest that the 90K protein associates non-covalently with the Mr 65,000 estrogen binding unit as a nonhormone binding component of the 9-10S receptor.     

Sarcoplasmic reticulum. X. The protein composition of sarcoplasmic reticulum membranes

The proteins of Sarcoplasmic reticulum membranes were resolved by polyacrylamide gel electrophoresis into several fractions ranging in mol wt from 300,000 to about 30,000. The ATPase enzyme involved in Ca²⁺ transport is associated with a major protein fraction and its molecular weight based on its electrophoretic mobility on polyacrylamide gels in the presence of sodium dodecylsulfate is about 106,000. Reducing agents (β-mercaptoethanol or dithiothreitol) cause the dissociation of membrane proteins into subunits of 20,000–60,000 mol wt, which can be separated by electrophoresis or Sephadex G-150 chromatography.     

The influence of Nematospiroides dubius on subsequent Nippostrongylus brasiliensis infections in mice.

The fecundity and longevity of Nippostrongylus brasiliensis was prolonged in mice previously infected with Nematospiroides dubius only when the former developed from the larval stage in those mice. Such worms appeared to be less immunogenic than worms which developed in mice never exposed to N. dubius. It is proposed that prolonged fecundity and longevity resulted from an adaptation undertaken by the worms in the face of host antibodies which had been developed against the pre-existing N. dubius infection.     

PHOSPHORYLATION OF ENDOGENOUS PROTEINS IN MYELIN OF RAT BRAIN

Abstract— The phosphorylation of endogenous proteins occurring in the myelin of rat brain was examined using the method of sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Two myelin basic proteins and at least five more proteins were phosphorylated after incubation of myelin fraction in the presence of ATP + Mg²⁺. The apparent molecular weights of the proteins other than the myelin basic proteins were 120,000, 76,000, 60,000, 41,000 and 38,000, respectively. The proteins of mol wt 60,000. 41,000 and 38,000 were extracted by treatment with hydrochloric acid, whereas those of mol wt 120,000 and 76,000 were insoluble in hydrochloric acid and chloroform-methanol. Folch-Lees proteolipid protein was not found to be phosphorylated under the conditions studied. The endogenous phosphorylation of the proteins was not stimulated by adenosine 3',5'-monophosphate.     

Identification of two intermediates during processing of profilaggrin to filaggrin in neonatal mouse epidermis

A major event in the keratinization of epidermis is the production of the histidine-rich protein filaggrin (26,000 mol wt) from its high molecular weight (greater than 350,000) phosphorylated precursor (profilaggrin). We have identified two nonphosphorylated intermediates (60,000 and 90,000 mol wt) in NaSCN extracts of epidermis from C57/Bl6 mice by in vivo pulse-chase studies. Results of peptide mapping using a two-dimensional technique suggest that these intermediates consist of either two or three copies of filaggrin domains. Each of the intermediates has been purified. The ratios of amino acids in the purified components are unusual and essentially identical. The data are discussed in terms of a precursor containing tandem repeats of similar domains. In vivo pulse-chase experiments demonstrate that the processing of the high molecular weight phosphorylated precursor involves dephosphorylation and proteolytic steps through three-domain and two-domain intermediates to filaggrin. These processing steps appear to occur as the cell goes through the transition cell stage to form a cornified cell.     

Cytochrome P-450 in culture forms of Trypanosoma cruzi

Trypanosoma cruzi epimastigote and trypomastigote forms contain microsomal peptides in the 40-60,000 mol. wt region, some of which are heme-staining-positive and are induced by phenobarbital, as indicated by SDS-gel electrophoresis and by double-labeling experiments. Epimastigotes show induced peptides of mol. wt 56,000, 52,000, 49,000, 44,000, 42,000 and 40,500 whereas only one peptide (52,500 mol. wt) is increased in trypomastigotes. Fractionation of microsomes derived from epimastigotes by octylamine Sepharose-4B column chromatography reveals the presence of two heme peptides with mol. wt of 55,800 and 56,600. The pooled fraction has a typical cytochrome P-450 CO-difference spectrum and appears to correspond to a high spin form. The demonstration of the existence of this family of hemoproteins in T. cruzi further supports the idea that resistance to chemotherapeutic agents is due to active metabolism. The active metabolism, however, may not be similar in the various developmental forms of this organism since differences exist in the patterns of induction of heme-positive microsomal peptides.     

Regulation of toxocariasis in mice selectively reared for high and low immune responses to Nematospiroides dubius

Test mice have been selectively reared for high (H) or low (L) immune responses to Nematospiroides dubius. After secondary infection with N. dubius, the L mice voided ten times as many eggs in their faeces as the H mice, and at necropsy, 71% versus 20% of the inoculum of N. dubius were recovered as adult worms from the L and H mice respectively. Furthermore, N. dubius were more fecund in the L than in H mice. High or low immune responsiveness was not restricted to N. dubius infection in these mice but was also observed during Toxocara canis infection. The migration of T. canis larvae from gut via the liver to skeletal muscle and CNS was inhibited in H versus L mice. Many more larvae were recovered from the livers of H compared with L mice which was indicative of greater immunity in the H mice. The protective immune response in H compared with L mice to both N. dubius and T. canis included pronounced eosinophilia and elevated antiparasite antibody titres.     

A comparison of the major surfactant-associated proteins in different species

We examined the major surfactant-associated proteins in a number of species by using two-dimensional gel electrophoresis, protein blotting and immunostaining. All species have a 30,000 to 35,000 mol. wt protein group consisting of multiple isoforms with isoelectric points ranging from pH 4.4 to 5.6. The techniques used in this study have resolved three component subgroups within the 35 K group. A group of proteins at 60,000-65,000 mol. wt has also been consistently identified. We conclude that remarkable similarity exists among the major surfactant-associated proteins from various mammals with regard to isoelectric points, molecular weights and antigenic sites.     

Identification of the subunit proteins of 10-nm neurofilaments isolated from axoplasm of squid and Myxicola giant axons

Neurofilaments were isolated from the axoplasm of the giant axons of Myxicola infundibulum and squid. The axoplasm was fractionated by discontinuous sucrose gradient centrifugation and gel filtration on Sepharose 4B. The fractions were monitored for neurofilaments by electron microscopy. When isolated in the presence of chelating agents, the neurofilaments of Myxicola are composed almost entirely of protein subunits with mol wt of 150,000 and 160,000. Squid neurofilaments contain two major proteins with mol wt of 200,000 and 60,000. These proteins are compared with other intermediate filament proteins which have been reported in the literature.