Flight muscle ultrastructure of susceptible and refractory mosquitoes parasitized by larval Brugia pahangi.

On parasitization with larval Brugia pahangi the infected flight muscle fibres of "resistant" Anopheles labranchiae atroparvus undergo the following ultrastructural changes. The fibres become almost totally devoid of glycogen, their sarcoplasmic reticulum becomes elongate and closely associated with muscle fibrils. These fibrils degenerate and vesicles appear both within the degenerate fibril and within elements of the sarcoplasmic reticulum. Vesicles accumulate around the worm and degenerate to a uniform mass which eventually becomes melanized from its inner edge (next to the parasite) outwards. The infected flight muscle fibres of both "resistant" Aedes aegypti and "susceptible" Aedes togoi are almost totally devoid of glycogen granules, but show no other ultrastructural change from the uninfected state.     

Incorporation of orally administered radioactive precursors into nucleic acids and ribosomes of housefly ovaries

After emergence female houseflies were fed for 4 days on a diet containing ¹⁴C-orotic acid and ³H-thymidine-5-triphosphate, or ³H-leucine. Nucleic acids and ribosomes were then isolated from the ovaries and studied by MAK column chromatography and sedimentation analysis respectively. The ultraviolet absorption and radioactivity of the fractions were also measured. After MAK column chromatography, the u.v. elution pattern showed that only tow distinct peaks, corresponding to tRNA and rRNA were present. A similar elution pattern was obtained by measuring the ¹⁴C from ¹⁴C-orotic acid incorporated into the RNA. Because of the small quantity present, DNA was not measurable by u.v. absorption, but by determining the incorporation of ³H from ³H-TTP, its presence was clearly evident.Sedimentation analysis of ovarian ribosomes revealed four polymeric forms besides subunits and monomers. The incorporation of ¹⁴C-orotic acid and ³H-leucine into the ribosomes was used to follow the synthesis of rRNA and rProtein respectively. Sucrose density gradient centrifugation of the rRNA indicated that the ovarian rRNA consisted primarily of 28 and 18 S particles.     

Biosynthesis of respiratory-tract mucins. Incorporation of radioactive precursors into glycoproteins by canine tracheal explants in vitro

1. Canine tracheal explants, cultured in medium 199, actively incorporated radioactive precursors into secreted macromolecules in vitro. 2. Puromycin, 6-diazo-5-oxo-l-norleucine and ouabain markedly inhibited the incorporation of these precursors. 3. Exogenous glucosamine at concentrations above 20mm caused a greater than 50% inhibition of the incorporation of l-[G-(3)H]fucose and l-[U-(14)C]serine. 4. Carbohydrate content of the purified secretions was approximately 50% and consisted principally of galactose, N-acetylglucosamine, N-acetylgalactosamine, fucose and sialic acids. 5. Chromatography on DEAE-cellulose and Bio-Gel A-150m and equilibrium density-gradient centrifugation in a CsCl gradient confirmed the presence of mucous glycoproteins. 6. Electrophoresis on 1% agarose gels gave profiles that were identical with canine respiratory mucus obtained in vivo. 7. These results support the utility of the explant system for studies of respiratory secretions.     

Defense reaction of mosquitoes to filarial worms: role of the microfilarial sheath in the response of mosquitoes to inoculated Brugia pahangi microfilariae

The melanization response of Aedes trivittatus and A. aegypti (black-eyed Liverpool strain) against intrathoracically inoculated sheathed and chemically exsheathed Brugia pahangi microfilariae (mff) was assessed daily through 5 days postinoculation (PI). Response of A. aegypti against exsheathed mff was significantly reduced on all days compared with the response against sheathed mff, and a significantly greater percentage of exsheathed mff were alive through 4 days PI than were sheathed mff. The melanization response of A. trivittatus was nearly 100% effective against either sheathed or exsheathed mff by Day 2 PI. When mff were allowed to migrate through A. aegypti midguts in vitro before inoculation into intact A. aegypti, nearly 94% ($\text{120}\text{128}$) of the parasites recovered had avoided the response and were developing. Penetration of A. trivittatus midguts in vitro by mff before inoculation into intact A. trivittatus did not prevent a melanization response. Inoculation of mff into A. trivittatus following A. aegypti midgut penetration, however, resulted in almost 60% ($\text{98}\text{171}$) of the mff avoiding the response and developing as normal L₁ larvae after 5 days PI. The possibility of mff acquiring host antigens during midgut penetration and therefore avoiding recognition as nonself by mosquitoes, and (or) the possibility of the midgut environment modifying or stimulating mff to inhibit the response of mosquitoes are discussed.     

Defense reactions of mosquitoes to filarial worms: comparative studies on the response of three different mosquitoes to inoculated Brugia pahangi and Dirofilaria immitis microfilariae

The melanization response of Aedes trivittatus and the Rockefeller (RKF) and black-eyed Liverpool (LVP) strains of Aedes aegypti against intrathoracically inoculated Dirofilaria immitis and Brugia pahangi microfilariae (mff) was investigated. All mff of either species were melanized in A. trivittatus following Day 2 postinoculation, and the response of this species was significantly more rapid and effective than either strain of A. aegypti. The refractory RKF strain had a significantly greater response against both D. immitis and B. pahangi than the highly susceptible LVP strain, but data suggest that the increased responsiveness was due to a physiologic incompatibility in RKF A. aegypti, thereby resulting in a greater mortality and subsequent melanization of inoculated mff. Inoculation of large numbers of mff overloaded the defense capabilities of A. aegypti (LVP), but not those of A. trivittatus. The melanization response against D. immitis mff was effectively reduced for up to 4 days in A. aegypti (LVP), but for only 1 day in A. trivittatus, when mosquitoes were maintained on a 0.3 m sucrose diet containing from 0.1 to 1.0% (w/v) phenylthiourea.     

Incorporation of radioactive precursors into glycosaminoglycans by rat muscle fibroblasts exposed to a solubilized rat bone matrix fraction

Confluent cultures of rat muscle fibroblastic cells respond by increased glycosaminoglycan (GAG) synthesis when cultured in medium containing a solubilized bone matrix fraction (SBM) at a concentration of 100 micrograms/ml. The metabolism of the GAG associated with the cell pellet, the cell surface and the tissue culture medium fractions was studied, in the presence and absence of SBM, by measuring the incorporation of radioactivity from [3H]glucosamine and [35S]SO4 into the isolated GAG. Net synthesis of hyaluronic acid and of chondroitin sulfate in the medium fraction increased more rapidly in cultures containing SBM compared to controls, and the accumulation of labelled GAG in the medium of the treated cultures was approximately linear with respect to the length of incubation. The addition of SBM also resulted in increased incorporation of 3H and of 35S into the GAG of the cell surface and cell pellet fractions. In these fractions, stimulation of incorporation of radioactivity occurred in two waves: an early, relatively minor increase and a later relatively major increase. The relatively major stimulation of radioactivity into the GAG of the cell surface fraction occurred between 24 and 48 h and was independent of any apparent effect of serum.     

Defense reactions of mosquitoes to filarial worms: potential mechanism for avoidance of the response by Brugia pahangi microfilariae

The melanization response of Aedes trivittatus and the black-eyed Liverpool (LVP) and Rocke-feller (RKF) strains of Aedes aegypti against intrathoracically inoculated Brugia pahangi microfilariae (mff) that previously had penetrated LVP, RKF, or A. trivittatus midguts in vitro was assessed at 1, 3, and 5 days postinoculation (PI). LVP and RKF midgut-derived mff almost totally avoided the melanization response and developed normally in LVP strain A. aegypti, and although over 90% of these mff died by 5 days PI in RKF mosquitoes, the majority of these were not melanized. A. aegypti midgut-derived mff also were able to avoid the response of A. trivittatus in 33–43% of the cases. Penetration through A. trivittatus midguts, however, did not significantly affect the ability of mff to avoid the melanization response in any of the mosquitoes examined. Allogeneic and xenogeneic tissue inplants were accepted by all three mosquito species examined. Data presented support the hypothesis that mff avoid immune recognition in compatible mosquitoes by coating themselves with midgut material(s) during penetration of the midgut in their migration to the hemocoel.