The fine structure of Culicinomyces clavisporus invading mosquito larvae

Electron microscope observations were made of the Australian and U.S. strains of Culicinomyces clavisporus infecting mosquito larvae. The wall of the conidium is composed of an inner (primary) layer, an outer (secondary) layer, and an exterior coating of a mucopolysaccharide substance believed responsible for conidial adhesion to the host cuticle prior to germination and penetration. In some instances the wall of the conidium is ruptured during germination and new wall layers and mucoid coating form around the germ tube whereas in other specimens the conidial wall layers extend around the germ tube without fracturing. The most common invasion site is through the larval foregut following ingestion of conidia. The apex of the germ tube presses tightly against the surface of the foregut cuticle and the mucilaginous coating is stripped away. There is evidence to suggest that the host epicuticle, which disappears across the zone of contact with the germ tube, is utilized for nutrition of the invading fungus. A collar of cuticle forms around the germ tube apex and a narrow penetrant hyphae extends into the procuticle. It is believed that cuticular penetration is primarily enzymatic assisted by mechanical pressure. The penetrant hypha swells into an oval cell in the hypodermal region and vegetative hypha then invade the hemocoel. The cells of the hypodermis develop signs of degeneration presumably due to the secretion of toxic substances from the invading hyphae. Host reactions, involving melanization of the host tissues, are sometimes evident among the invading penetrant hyphae in the cuticle or in the hypodermal cells in contact with the fungus. Melanized capsules form around some of the hyphae within the hemocoel. These latter reactions do not directly involve host blood cells and are examples of “humoral encapsulation” similar to that described by other authors during invasion of pathogenic organisms into mosquito larvae and chironomid larvae.     

Comparative visual acuity of coleoid cephalopods

The pelagic realm of the ocean is characterized by extremelyclear water and a lack of surfaces. Adaptations to the visualecology of this environment include transparency, fluorescence,bioluminescence, and deep red or black pigmentation. While thesignals that pelagic organisms send are increasingly well-understood,the optical capabilities of their viewers, especially for predatorswith camera-like vision such as fish and squid, are almost unknown.Aquatic camera-like vision is characterized by a spherical lensfocusing an image on the retina. Here, we measured the resolvingpower of the lenses of eight species of pelagic cephalopodsto obtain an approximation of their visual capabilities. Wedid this by focusing a standard resolution target through dissectedlenses and calculating their modulation transfer functions.The modulation transfer function (MTF) is the single most completeexpression of the resolving capabilities of a lens. Since theoptical and retinal capabilities of an eye are generally well-matched,we considered our measurements of cephalopod lens MTF to bea good proxy for their visual capabilities in vivo. In general,squid have optical capabilities comparable to other organismsgenerally assumed to have good vision, such as fish and birds.Surprisingly, the optical capability of the eye of Vampyroteuthisinfernalis rivals that of humans.     

Synthesis and analysis of the enantiomers of calmidazolium,and a 1H NMR demonstration of a chiral interaction with calmodulin

Calmidazolium {R24571, 1-[bis(4-chlorophenyl)methyl]-3-[2-(2,4-dichlorophenyl)-2-[(2,4-dichlorophenyl)methoxy]ethyl]-1H-imidazolium chloride} is a potent calmodulin inhibitor. This paper describes the synthesis and properties of the enantiomers of calmidazolium from the enantiomers of miconazole {1(N)-(2-(2,4-dichlorobenzyloxy)-2-(2,4-dichlorophenyl))-ethyl imidazole}, prepared from the racemate by chiral preparative scale high performance liquid chromatography. Overlap between ligand and protein resonances in the aromatic region of the ¹H NMR spectrum of the calmidazolium-calmodulin complexes has been obviated by preparation of the protein with all of its nine phenylalanine rings deuterated (Phe-d₅ calmodulin). This has been accomplished by the overexpression of calmodulin derived from Trypanosoma brucei rhodiesiense in E. coli in a medium supplemented with ring-deuterated phenylalanine. The kinetics of binding of each enantiomer are slow on the ¹H NMR time scale as judged by the behaviour of the H2 resonance of Histidine-107, which is clearly visible under the sample conditions used. The aromatic spectral regions of the protein-bound (+) and (−) enantiomers contrast strikingly, reflecting differences in bound environment and/or conformation. Chirality 8:545–500, 1996. © 1997 Wiley-Liss, Inc.     

A theoretical expression for the protection associated with stoichiometric and catalytic scavengers in a single compartment model of organophosphorus poisoning

The ability of certain organophosphorus (OP) compounds to inhibit acetylcholinesterase (AChE) has made them useful for industrial (insecticides) and military (nerve agents) purposes. We have previously published a single compartment mathematical model of the interactions between OP nerve agents and the enzymes affected by these agents. That model, which could be used to predict the LD50 of seven nerve agents in rats, has been extended to include the protective actions of stoichiometric and catalytic OP-scavenger enzymes (delivered as pretreatments) so that protective ratios attributable to the scavengers may be predicted. Prediction of expected human protection from in vitro rate constant and initial enzyme level measurements is the ultimate goal for this work. The enhanced model predicts the LD50 from rate constants of the OP agent's binding reactions with AChE, carboxylesterase (CaE) and a stoichiometric scavenger (S); a first-order OP elimination rate (including a contribution due to a catalytic scavenger); and whole body estimates of AChE, CaE and S. The ratio of the scavenger-treated LD50 estimate to the scavenger-free LD50 estimate provided a theoretical expression describing the scavenger's contributions to the protective ratio. Published in vivo protective ratios for two stoichiometric scavengers (fetal bovine serum AChE and human utyrylcholinesterase) against challenge by several OP agents in mice were compared with ratios predicted by the model. A linear regression analysis of in vivo protective ratios in mice versus the ratios predicted by the model from the in vitro measurements resulted in an R(2) value of 0.902. The catalytic scavenger portion of the theory could not be validated due to a lack of published data. We conclude that the one-compartment model can be used to make reasonable estimates of the protective ratio attributable to stoichiometric scavengers, but can make no conclusions regarding the ability of the model to predict catalytic scavenger protection ratios.     

Targeted expression of tetanus neurotoxin interferes with behavioral responses to sensory input in Drosophila.

Targeted inactivation of neurons by expression of toxic gene products is a useful tool to assign behavioral functions to specific neurons or brain structures. Of a variety of toxic gene products tested, tetanus neurotoxin light chain (TNT) has the least severe side effects and can completely block chemical synapses. By using the GAL4 system to drive TNT expression in a subset of chemo- and mechanosensory neurons, we detected walking and flight defects consistent with blocking of relevant sensory information. We also found, for the first time, an olfactory behavioral phenotype associated with blocking of a specific subset of antennal chemoreceptors. Similar behavioral experiments with GAL4 lines expressing in different subsets of antennal chemoreceptors should contribute to an understanding of olfactory coding in Drosophila. To increase the utility of the GAL4 system for such purposes, we have designed an inducible system that allows us to circumvent lethality caused by TNT expression during early development.     

FREEZE-FRACTURED CHLOROPLAST MEMBRANES OF GONYAULAX POLYEDRA (PYRROPHYTA)1

The chloroplast membranes of Gonyaulax polyedra Stein were studied in replicas of rapidly frozen and fractured cells. The thylakoid EF_s face lacked the large 15–16 nm particles characteristic of plants with the light-harvesting chlorophyll a/b protein, presumably because the principal light-harvesting protein of Gonyaulax is the small water-soluble peridinin-chlorophyll-protein and the chlorophyll a/b protein is absent. As in other plants, the EF_s thylakoid fracture face carried more particles (4 ×) than EF_uface. The PF faces of the thylakoid showed twice as many particles as did the EF_s faces. No circadian differences in the number or size of thylakoid membrane particles could be detected. Three membranes comprise the chloroplast envelope in Gonyaulax. They could be clearly differentiated in freeze-fractured cells. The middle envelope membrane carried many fewer particles on both the EF and PF faces than did the other two envelope membranes. The PF faces of both the outer and inner envelope membranes showed more particles than the EF faces, as do many other membranes which have been examined.     

Effect of lactation stage and concurrent pregnancy on milk composition in the bottlenose dolphin

Although many toothed whales (Cetacea: Odontoceti) lactate for 2–3 years or more, it is not known whether milk composition is affected by lactation stage in any odontocete species. We collected 64 pooled milk samples spanning 1–30 months postpartum from three captive bottlenose dolphins Tursiops truncatus . Milks were assayed for water, fat, crude protein (TN × 6.38) and sugar; gross energy was calculated. Ovulation and pregnancy were determined via monitoring of milk progesterone. Based on analysis of changes in milk composition for each individual dolphin, there were significant increases ( P <0.05) in fat (in all three dolphins) and crude protein (in two of three), and a decrease ( P <0.05) in water (in two of three) over the course of lactation, but the sugar content did not change. In all three animals, the energy content was positively correlated with month of lactation, but the percentage of energy provided by crude protein declined slightly but significantly ( P <0.05). At mid-lactation (7–12 months postpartum, n =17), milk averaged 73.0±1.0% water, 12.8±1.0% fat, 8.9±0.5% crude protein, 1.0±0.1% sugar, 1.76±0.09 kcal g⁻¹ (=7.25 kJ g⁻¹) and 30.3±1.3% protein:energy per cent. This protein:energy per cent was surprisingly high compared with other cetaceans and in relation to the growth rates of calves. Milk progesterone indicated that dolphins ovulated and conceived between 413 and 673 days postpartum, following an increase in milk energy density. The significance of these observed compositional changes to calf nutrition will depend on the amounts of milk produced at different stages of lactation, and how milk composition and yield are influenced by sampling procedure, maternal diet and maternal condition, none of which are known.     

Binding of non-natural 3'-nucleotides to ribonuclease A

2'-Fluoro-2'-deoxyuridine 3'-phosphate (dU(F)MP) and arabinouridine 3'-phosphate (araUMP) have non-natural furanose rings. dU(F)MP and araUMP were prepared by chemical synthesis and found to have three- to sevenfold higher affinity than uridine 3'-phosphate (3'-UMP) or 2'-deoxyuridine 3'-phosphate (dUMP) for ribonuclease A (RNase A). These differences probably arise (in part) from the phosphoryl groups of 3'-UMP, dU(F)MP, and araUMP (pK(a) = 5.9) being more anionic than that of dUMP (pK(a) = 6.3). The three-dimensional structures of the crystalline complexes of RNase A with dUMP, dU(F)MP and araUMP were determined at < 1.7 A resolution by X-ray diffraction analysis. In these three structures, the uracil nucleobases and phosphoryl groups bind to the enzyme in a nearly identical position. Unlike 3'-UMP and dU(F)MP, dUMP and araUMP bind with their furanose rings in the preferred pucker. In the RNase A.araUMP complex, the 2'-hydroxyl group is exposed to the solvent. All four 3'-nucleotides bind more tightly to wild-type RNase A than to its T45G variant, which lacks the residue that interacts most closely with the uracil nucleobase. These findings illuminate in atomic detail the interaction of RNase A and 3'-nucleotides, and indicate that non-natural furanose rings can serve as the basis for more potent inhibitors of catalysis by RNase A.