Hemolymph-dependent and -independent responses in Drosophila immune tissue

Insects possess an antimicrobial defense response that is similar to the mammalian innate immune response. The innate immune system is designed to recognize conserved components of microorganisms called pathogen-associated molecular patterns (PAMPs). How host receptors detect PAMPs and transmit the signals to mount the immune response is being elucidated. Using GFP-Dorsal, -Dif, and -Relish reporter proteins in ex vivo assays, we demonstrate that Drosophila fat bodies, a major immune tissue, have both hemolymph-dependent and -independent responses. Microbial preparations such as lipoteichoic acid (LTA) and peptidoglycan (PGN) can stimulate some responses from dissected and rinsed larval fat bodies. Therefore, at least some aspects of recognition can occur on fat body cell surfaces, bypassing the requirement of hemolymph. Our results also show that supernatants from bacterial cultures can stimulate the nuclear translocation of Dorsal in dissected fat bodies, but this stimulation is strictly hemolymph-dependent. Various biochemical assays suggest that the factors from bacterial supernatants that stimulate the hemolymph-dependent nuclear translocation are likely made up of proteins. We further show that Dorsal mutant larvae have much lower phenoloxidase activity, consistent with a more important role of Dorsal in innate immunity than previously shown.     

Ventricle and outflow tract of the African lungfish Protopterus dolloi

We report a morphologic study of the heart ventricle and outflow tract of the African lungfish Protopterus dolloi. The ventricle is saccular and appears attached to the anterior pericardial wall by a thick tendon. An incomplete septum divides the ventricle into two chambers. Both the free ventricular wall and the incomplete ventricular septum are entirely trabeculated. Only a thin rim of myocardium separates the trabecular system from the subepicardial space. The outflow tract consists of proximal, middle, and distal portions, separated by two flexures, proximal and distal. The proximal outflow tract portion is endowed with a layer of compact, well-vascularized myocardium. This portion is homologous to the conus arteriosus observed in the heart of most vertebrates. The middle and distal outflow tract portions are arterial-like, thus being homologous to the bulbus arteriosus. However, the separation between the muscular and arterial portions of the outflow tract is not complete in the lungfish. A thin layer of myocardium covers the arterial tissue, and a thin layer of elastic tissue underlies the conus myocardium. Two unequal ridges composed of loose connective tissue, the spiral and bulbar folds, run the length of the outflow tract. They form an incomplete division of the outflow tract, but fuse at the distal end. The two folds are covered by endocardium and contain collagen, elastin, and fibroblast-like cells. They appear to be homologous to the dextro-dorsal and sinistro-ventral ridges observed during the development of the avian and mammalian heart. Two to three rows of vestigial arterial-like valves appear in the dorsal and ventral aspects of the conus. These valves are unlikely to have a functional role. The possible functional significance of the "gubernaculum cordis," the thick tendon extending between the anterior ventricular surface and the pericardium, is discussed.     

The interplay of increased urea synthesis and reduced ammonia production in the African lungfish Protopterus aethiopicus during 46 days of aestivation in a mucus cocoon

This study was undertaken to test the hypothesis that the rate of urea synthesis in Protopterus aethiopicus was up-regulated to detoxify ammonia during the initial phase of aestivation in air (day 1-day 12), and that a profound suppression of ammonia production occurred at a later phase of aestivation (day 35-day 46) which eliminated the need to sustain the increased rate of urea synthesis. Fasting apparently led to a greater rate of nitrogenous waste excretion in P. aethiopicus in water, which is an indication of increases in production of endogenous ammonia and urea probably as a result of increased proteolysis and amino acid catabolism for energy production. However, 46 days of fasting had no significant effects on the ammonia or urea contents in the muscle, liver, plasma and brain. In contrast, there were significant decreases in the muscle ammonia content in fish after 12, 34 or 46 days of aestivation in air when compared with fish fasting in water. Ammonia was apparently detoxified to urea because urea contents in the muscle, liver, plasma and brain of P. aethiopicus aestivated for 12, 34 or 46 days were significantly greater than the corresponding fasting control; the greatest increases in urea contents occurred during the initial 12 days. There were also significant increases in activities of some of the hepatic ornithine-urea cycle enzymes from fish aestivated for 12 or 46 days. Therefore, contrary to a previous report on P. aethiopicus, our results demonstrated an increase in the estimated rate of urea synthesis (2.8-fold greater than the day 0 fish) in this lungfish during the initial 12 days of aestivation. However, the estimated rate of urea synthesis decreased significantly during the next 34 days. Between day 35 and day 46 (12 days), urea synthesis apparently decreased to 42% of the day 0 control value, and this is the first report of such a phenomenon in African lungfish undergoing aestivation. On the other hand, the estimated rate of ammonia production in P. aethiopicus increased slightly (14.7%) during the initial 12 days of aestivation as compared with that in the day 0 fish. By contrast, the estimated rate of ammonia production decreased by 84% during the final 12 days of aestivation (day 35-day 46) compared with the day 0 value. Therefore, it can be concluded that P. aethiopicus depended mainly on increased urea synthesis to ameliorate ammonia toxicity during the initial phase of aestivation, but during prolonged aestivation, it suppressed ammonia production profoundly, eliminating the need to increase urea synthesis which is energy-intensive.     

Prolactin and epidermal growth factor regulation of the proliferation,morphogenesis, and functional differentiation of normal rat mammary epithelial cells in three dimensional primary culture

The epithelial cell-specific effects of prolactin and epidermal growth factor (EGF) on the development of normal rat mammary epithelial cells (MEC) were evaluated using a three dimensional primary culture model developed in our laboratory. Non-milk-producing MEC were isolated as spherical end bud-like mammary epithelial organoids (MEO) from pubescent virgin female rats. The cultured MEO developed into elaborate multilobular and lobuloductal alveolar organoids composed of cytologically and functionally differentiated MEC. Prolactin (0.01–10 μg/ml) and EGF (1–100 ng/ml) were each required for induction of cell growth, extensive alveolar, as well as multilobular branching morphogenesis, and casein accumulation. MEO cultured without prolactin for 14 days remained sensitive to the mitogenic, morphogenic, and lactogenic effects of prolactin upon subsequent exposure. Similarly, cells cultured in the absence of EGF remained sensitive to the mitogenic and lactogenic effects of EGF, but were less responsive to its morphogenic effects when it was added on day 14 of a 21-day culture period. If exposure to prolactin was terminated after the first week, the magnitude of the mitogenic and lactogenic effects, but not the morphogenic response was decreased. Removal of EGF on day 7 also reduced the mitogenic response, but did not have any effect on the magnitude of the lactogenic or morphogenic responses. These studies demonstrate that physiologically relevant development of normal MEC can be induced in culture and that this model system can be used to study the mechanisms by which prolactin and EGF regulate the complex developmental pathways operative in the mammary gland. © 1995 Wiley-Liss, Inc.     

Prooxidant and antioxidant effects of trolox on ferric ion-induced oxidation of erythrocyte membrane lipids

Achatinin_H (ATN_H)is a lectin, isolated from the hemolymph ofAchatina fulica snail, which has been shown to have narrow specificity towards 9-O-acetyl sialic acid. Usually ATN_H does not agglutinate normal human erythrocytes, however, it is capable of agglutinating erythrocytes of patients suffering from acute lymphocytic and acute myelogenous leukemia. Determination of binding constants, numbers of binding sites and lectin overlay experiments using patients' erythrocytes ghost, have suggested that some alterations in erythrocyte cell surface sialoglycoproteins or more precisely appearance of some O-acetylated sialoglycoprotein as a result of pathological transformations has caused this change in the binding of ATN_H.Abbreviations ATN_H Achatinin_H - 9-OAc-NeuAc 9-O-acetyl N-acetyl neuraminic acid - BSM Bovine submaxillary mucin - TBS Tris-buffered saline - SDS-PAGE Sodium dodecyl sulphate polyacrylamide gel electrophoresis - BSA Bovine serum albumin - HA Hemagglutination assay - ALL Acute lymphocytic leukemia - AML Acute myelogenuos leukemia - NP 40 Nonidet 40     

The colorful mantle of the giant clam,Tridacna squamosa,expresses a light-dependent manganese superoxide dismutase to ameliorate oxidative stresses due to its symbiotic association with zooxanthellae

Coral Reefs - Giant clams harbor extracellular symbiotic zooxanthellae in a tubular system that pervades mainly the fleshy and colorful outer mantle. During insolation, the symbiotic zooxanthellae...     

Modulation of desmin intermediate filament assembly by a monoclonal antibody

We have used a monoclonal antibody against desmin to examine the assembly of intermediate filaments (IF) from their building blocks, the tetrameric protofilaments. The antibody, designated D76, does not cross react with any other IF proteins (Danto, S.I., and D.A. Fischman. 1984. J. Cell Biol. 98:2179-2191). It binds to a region amino-terminal to cys-324 of avian desmin that is resistant to chymotrypsin and trypsin digestion, and in the electron microscope appears to bind to the ends of tetrameric protofilaments. In combination, these findings suggest that the epitope of the antibody resides at the amino-terminal end of the alpha-helical rod domain. Preincubation of desmin protofilaments with an excess of D76 antibodies blocks their subsequent assembly into IF. In the presence of sub-stoichiometric amounts of antibodies, IF are assembled from protofilaments but they are morphologically aberrant in that (a) they are capped by IgG molecules at one or both ends; (b) they are unraveled to varying degree, revealing a characteristic right-handed helical arrangement of sub-filamentous strands of different diameters. The antibody binds only to the ends but not along the length of desmin IF. The most straightforward explanation for this is that the epitope resides in a part of the desmin molecule that becomes buried within the core of the filament upon polymerization and is therefore inaccessible to the antibody.     

Comparative effects of antioxidants on enzymes involved in glutathione metabolism

The present study was designed to examine changes in glutathione metabolism in the liver of mice as influenced by supplementation of their diet with 1 of 4 antioxidants: butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), vitamin E and selenium. In addition to determination of the acid-soluble thiol levels, 5 different enzymes involved with glutathione utilization and synthesis were measured: glutathione transferase, γ-glutamyl transpeptidase, selenium-dependent glutathione peroxidase, γ-glutamylcysteine synthetase and glutathione reductase. All 4 antioxidants produced significant increases in glutathione transferase activity, with BHA and BHT being much more effective than the other two. With the exception of vitamin E, BHA, BHT and selenium all resulted in a slight enhancement in the activity of glutathione reductase as well as in the acid-soluble thiol level. On the other hand, the induction of γ-glutamyl transpeptidase and γ-glutamylcysteine synthetase was responsive to only vitamin E and selenium supplementation, respectively. Although the influence of each of these antioxidants in glutathione metabolism appears to be specific and somewhat compartmentalized, the overall impression is that of an increased capacity for glutathione-conjugate formation and recovery of reduced glutathione. These biochemical changes in glutathione metabolism may be relevant to the anticarcinogenic effects observed with BHA, BHT and selenium.