Induction of blindness by formoguanamine hydrochloride in adult male roseringed parakeets (Psittacula krameri)

Formoguanamine (2,4-diamino-s-triazine) was known to be an effective chemical agent in inducing blindness in poultry chicks, but not in adult birds. The present study was undertaken to demonstrate the influences, if any, of this chemical on the visual performance and retinal histology in an adult sub-tropical wild bird, the roseringed parakeet (Psittacula krameri). Formoguanamine (FG) hydrochloride was subcutaneously injected into adult parakeets at a dosage of 25 mg (dissolved in 0.75 ml physiological saline)/100 g body weight/day, for two consecutive days while the control birds were injected only with a placebo. The effects were studied after 10, 20 and 30 days of the last treatment of FG. Within 24 h of the treatment of FG, about 90% of the total birds exhibited lack of visual responses to any light stimulus and even absence of pupillary light reactions. The remaining birds became totally blind on the day following the last injection of FG and remained so till the end of investigation. At the microscopic level, conspicuous degenerative changes were noted in the outer pigmented epithelium and the photoreceptive layer of rods and cones in the retinas of FG treated birds. A significant reduction in the thickness of the outer nuclear layer was also found in the retinas of FG treated parakeets, compared to that in the control birds. However, the inner cell layers of the retina in the control and FG administered parakeets were almost identical. It deserves special mention that the effects of FG, noted after 30 days of last treatment, were not very different from those noted just after 10 days of treatment. Collectively, the results of the present investigation demonstrate that FG can be used as a potent pharmacological agent for inducing irreversible blindness through selective damage in retinal tissue even in the adult wild bird, thereby making FG treatment an alternative euthanasic device to a cumbersome, stressful, surgical method of enucleation of the ocular system for laboratory studies.     

Synthesis and enzyme-catalyzed hydrolysis of a radical-masked glycosylated spin-label reagent

N1-Acetoxy-2,2,6,6-tetramethylpiperidin-4-yl 2,3,4,6-tetra-O-benzyl-alpha- and -beta-D-glucopyranosides (3-alpha, beta) and N1-acetoxy-2,2,5,5-tetramethylpyrrolin-3-oyl 2,3,4,6-tetra-O-benzyl-alpha- and -beta-D-glucopyranosylamines (9-alpha, beta) were synthesized in good yield by Schmidt's glycosylation method. Their subsequent O-debenzylation was proceeded successfully to give the desired products 1-alpha, and 1-beta in good yield, and 2-alpha in a low yield, without 2-beta by only short-timed hydrogenolysis in the presence of palladium-on-carbon (Pd-C) in a CHCl3-MeOH solvent system that included concentrated HCl. Upon enzyme-catalyzed hydrolysis, only 2-alpha was hydrolyzed by the esterase, while both of 1-alpha and 1-beta were not hydrolyzed by any other enzyme such as lipase. These 2-alpha can likely be used as a new water-soluble radical-masked glycosylated spin-label reagent.     

Synthesis of sialoglycopolypeptide for potentially blocking influenza virus infection using a rat α2,6-sialyltransferase expressed in BmNPV bacmid-injected silkworm larvae

Background

Interleukin-10 (IL-10) is a potent anti-inflammatory cytokine, with therapeutic applications in several autoimmune and inflammatory diseases. Oral administration of this cytokine alone, or in combination with disease-associated autoantigens could confer protection form the onset of a specific autoimmune disease through the induction of oral tolerance. Transgenic plants are attractive systems for production of therapeutic proteins because of the ability to do large scale-up at low cost, and the low maintenance requirements. They are highly amenable to oral administration and could become effective delivery systems without extensive protein purification. We investigated the ability of tobacco plants to produce high levels of biologically-active viral and murine IL-10.

Results

Three different subcellular targeting strategies were assessed in transient expression experiments, and stable transgenic tobacco plants were generated with the constructs that yielded the highest accumulation levels by targeting the recombinant proteins to the endoplasmic reticulum. The best yields using this strategy in T₁ plants were 10.8 and 37.0 μg/g fresh leaf weight for viral and murine IL-10, respectively. The recombinant proteins were purified from transgenic leaf material and characterized in terms of their N-glycan composition, dimerization and biological activity in in vitro assays. Both molecules formed stable dimers, were able to activate the IL-10 signaling pathway and to induce specific anti-inflammatory responses in mouse J774 macrophage cells.

Conclusion

Tobacco plants are able to correctly process viral and murine IL-10 into biologically active dimers, therefore representing a suitable platform for the production for these cytokines. The accumulation levels obtained are high enough to allow delivery of an immunologically relevant dose of IL-10 in a reasonable amount of leaf material, without extensive purification. This study paves the way to performing feeding studies in mouse models of autoimmune diseases, that will allow the evaluation the immunomodulatory properties and effectiveness of the viral IL-10 in inducing oral tolerance compared to the murine protein.     

A game model for the daily activity schedule of the male butterfly

A game model is developed of the daily schedule of matesearching activity by male butterflies, assuming that each male maximizes his expected mating success given a limited total time for mate search. The model predicts that (1) in the early morning, no male is active even though many females are emerging; (2) at a critical time, many males suddenly become active; and (3) the male's maximum activity occurs after the peak female emergence and before the peak capture efficiency. The inverse problem is also analyzed, in which the temporal pattern of capture efficiency is estimated from the knowledge of male activity and female emergence, assuming the evolutionarily stable strategy (ESS) condition. The model is then applied to data from a cabbage white butterfly (Pieris rapae crucivora) population and predicts that (1) females remain unmated for several hours on average after emergence, and (2) the male 's capture efficiency is rather low and increases significantly with time during the morning.     

Arginine-specific ADP-ribosyltransferase and its acceptor protein p33 in chicken polymorphonuclear cells: co-localization in the cell granules, partial characterization, and in situ mono(ADP-ribosyl)ation.

We have reported the purification and characterization of arginine-specific ADP-ribosyltransferase from hen liver nuclei [Tanigawa, Y. et al. (1984) J. Biol. Chem. 259, 2022-2029] and the DNA-dependent mono(ADP-ribosyl)ation of p33, an acceptor protein in the nuclei [Mishima, K. et al. (1989) Eur. J. Biochem. 179, 267-273]. In the present study, we obtained evidence that among various tissues and cells from chicken, polymorphonuclear cells, so-called heterophils, possess both the ADP-ribosyltransferase and p33 at high levels. Percoll density gradient centrifugation of the postnuclear fraction of the heterophils revealed the co-localization of ADP-ribosyltransferase with p33 in the granule fraction. The enzyme and p33 were purified approximately 219- and 3.77-fold, respectively, from postnuclear pellet fraction to apparent homogeneity. The properties of heterophil ADP-ribosyltransferase and p33 were compared with those of the liver enzyme and p33. The molecular mass of the heterophil enzyme was estimated by SDS-polyacrylamide gel electrophoresis to be 27.5 kDa. The enzyme activity was stimulated by a sulfhydryl agent and inhibited by lysolecithin, NaCl, and inorganic phosphate. The mono(ADP-ribosyl)ation of p33 was markedly enhanced by polyanion, such as DNA, RNA, or poly(L-glutamate). SDS-polyacrylamide gel electrophoretic analysis after limited trypsin proteolysis of p33s, purified from chicken heterophils and liver, showed much the same pattern. Thus, it appears that ADP-ribosyltransferase and p33 present in heterophils are identical to those in the liver, respectively. p33 is considered to be an in situ substrate for ADP-ribosyltransferase, since it was specifically mono(ADP-ribosyl)ated in permeabilized heterophils.(ABSTRACT TRUNCATED AT 250 WORDS)     

Site-directed mutagenesis of the cell-binding domain of human fibronectin: separable, synergistic sites mediate adhesive function

Polypeptide sequences required for function of the cell-binding domain of human fibronectin were analyzed by site-directed mutagenesis. Site-specific deletion of the putative recognition sequence Arg-Gly-Asp-Ser or an Asp-to-Glu mutation decreased the adhesive activity of fibronectin fusion proteins expressed in E. coli by greater than or equal to 97%. A second functional site over 0.5 kb away was identified by deletion mutagenesis. These mutants also showed a greater than or equal to 96% loss of activity, indicating cooperativity between sites. The two classes of mutant protein displayed synergism of activity in a trans complementation assay. Effective actin microfilament bundle organization was also dependent on the combined function of both sites. Thus, fibroblast adhesion and intracellular response to the fibronectin cell-binding domain involve two synergistic sites, each of major quantitative importance.     

Arginine-specific ADP-ribosyltransferase from rabbit skeletal muscle sarcoplasmic reticulum is solubilized as the active form with trypsin: partial purification and characterization

Arginine-specific ADP-ribosyltransferase from rabbit skeletal muscle sarcoplasmic reticulum was solubilized as the active form with trypsin. The enzyme was partially purified by subsequent chromatography, successively on DE-52, Con A-Sepharose and Sephadex G-75. An approximately 2,000-fold purification was achieved from the 105,000 x g supernatant of trypsin-treated membrane with a recovery of 2.8%. Dithiothreitol, which activates hen liver nuclear ADP-ribosyltransferase, inhibited the enzyme.     

Morphologic, morphometrical, and histological aspects of the eggs of four species of the genera Meccus (Hemiptera, Reduviidae, Triatominae)

The objective of this work was to analyze the morphological, morphometrical, and histological characteristics of eggs of four triatomine bugs species still not studied, in order to understand phylogenetic aspects and to facilitate parameters used in taxonomy, with the purpose of a specific and generic characterization of these vectors in public health. The eggs of each species had come from the collections of the Laboratory of Triatomine bugs and Culicid mosquitoes of the Faculdade de Saúde Pública - USP (Faculty of Public Health/USP). The morphologic studies were carried out through optic microscopy (OM) and scanning electronic microscopy (SEM). The eggs were measured with the help of the digital paquimeter Starrett 727. In relation to histological measures, Microtome Leica RM 2145 was used. Similarities were evidenced in the exocorial architecture of the eggs when they were studied by OM and SEM. This similarity among the eggs suggests a recent speciation, probably derived from common ancestry, representing a monophyletic group. Some structures were also detected by the histological cuts. The study has contributed to the magnification and recognition in generic and specific terms of the Triatominae subfamily. These new data will be able to subsidize a better understanding to determine roles for each vector species and to facilitate parameters to be used in taxonomy.