The immunocytochemical localization of superoxide dismutase in the enterocytes of the avian intestine: The effect of vitamin D3

Summary Both light microscopical and electron microscopical immunocytochemical techniques were utilized to localize CuZnsuperoxide dismutase (SOD) in the duodenum of normal, rachitic and vitamin-D₃-replete chicks. This enzyme catalyses the dismutation of the superoxide anion, a toxic free radical generated during the normal aerobic metabolism of most respiring cells. Light microscopy showed no SOD activity associated with the duodenal enterocytes of normal and rachitic chicks. However, in rachitic animals subsequently treated with vitamin D, i.e. vitamin-D-replete chicks, intense immunoreactivity for the enzyme was seen in association with the apical border of the duodenal absorptive cells. Immunostaining for SOD was not seen in goblet cells. With electron microscopy, immunostaining for SOD activity was identified in association with the apical microvilli and, to a lesser degree, with the terminal web, a well as in association with both lysosomes and peroxisomes. From this report it appears that there is a physiological relationship between vitamin D, SOD and the intestinal absorptive cell. However, the precise relationship must await further clarification.     

Photo-bleaching and photon saturation in flow cytometry.

In flow cytometry, small particles travel at a high speed through a bright light spot. The high light intensity at the point of measurement causes measurable photon saturation. This observation indicates that the rate at which individual dye molecules emit photons is close to the maximum emission rate. Despite the short exposure time, individual molecules may go through a few hundred excitation cycles while they are in the light beam. The absorbed light dose causes significant dye destruction. This article presents experimental procedures to determine the extent of photon saturation and photo-bleaching of dyes bound to cell nuclei in a flow cytometer. Measurements of Hoechst and propidium iodide bound to chromatin show that the amount of dye bleached per emitted photon is the same at low and high illumination intensities. This finding indicates that photon emission and dye destruction are both the result of the absorption of single excitation photons. The experimental observations allow rough estimates of the lifetime of the excited state and the lifetime of the molecule. The lifetime of the Hoechst 33258 bound to DNA is estimated to be 100 excitation-relaxation cycles. The average propidium iodide molecule lasts approximately 200 excitation-relaxation cycles. The theoretical considerations show that the optimal illumination conditions are different for bleaching and nonbleaching dyes. An optical arrangement for high precision measurements of bleaching dyes is presented.     

Effects of experimentally altered glutathione levels on life span, metabolic rate, superoxide dismutase, catalase and inorganic peroxides in the adult housefly, Musca domestica

Effects of varied levels of glutathione, an intracellular redox buffer, were examined in the adult male housefly in order to study the inter-relationship between enzymic and non-enzymic antioxidant defenses. An increase of over 100% in the concentrations of glutathione was induced by the administration of 3 mM L-2-oxothiazolidine-4-carboxylate (LOC), which increases the intracellular level of cysteine. A decrease in glutathione concentration of up to 85% was achieved by the administration of L-buthionine-SR-sulfoximine (BUS), which irreversibly inhibits glutamylcysteine synthetase. Life spans of houseflies were shortened by a decrease in the glutathione concentration, but were not prolonged by augmentation of glutathione. Metabolic rate and superoxide dismutase activity were independent of glutathione concentration. H2O2 was increased by both experimental regimes, whereas catalase activity was decreased by BUS. Results suggest that catalase activity is influenced by glutathione concentration.     

Localization of sequences regulating ancestral and acquired sites of esterase6 activity in Drosophila melanogaster

We have broadly defined the DNA regions regulating esterase6 activity in several life stages and tissue types of D. melanogaster using P- element-mediated transformation of constructs that contain the esterase6 coding region and deletions or substitutions in 5' or 3' flanking DNA. Hemolymph is a conserved ancestral site of EST6 activity in Drosophila and the primary sequences regulating its activity lie between -171 and -25 bp relative to the translation initiation site: deletion of these sequences decrease activity approximately 20-fold. Hemolymph activity is also modulated by four other DNA regions, three of which lie 5' and one of which lies 3' of the coding region. Of these, two have positive and two have negative effects, each of approximately twofold. Esterase6 activity is present also in two male reproductive tract tissues; the ejaculatory bulb, which is another ancestral activity site, and the ejaculatory duct, which is a recently acquired site within the melanogaster species subgroup. Activities in these tissues are at least in part independently regulated: activity in the ejaculatory bulb is conferred by sequences between -273 and -172 bp (threefold decrease when deleted), while activity in the ejaculatory duct is conferred by more distal sequences between -844 and -614 bp (fourfold decrease when deleted). The reproductive tract activity is further modulated by two additional DNA regions, one in 5' DNA (-613 to -284 bp; threefold decrease when deleted) and the other in 3' DNA (+1860 to +2731 bp; threefold decrease when deleted) that probably overlaps the adjacent esteraseP gene. Collating these data with previous studies suggests that expression of EST6 in the ancestral sites is mainly regulated by conserved proximal sequences while more variable distal sequences regulate expression in the acquired ejaculatory duct site.      

Isolation and partial characterization of Borrelia burgdorferi inner and outer membranes by using isopycnic centrifugation.

In order to characterize the protein composition of the outer membrane of Borrelia burgdorferi, we have isolated inner and outer membranes by using discontinuous sucrose density step gradients. Outer and inner membrane fractions isolated by this method contained less than 1 and 2%, respectively, of the total lactate dehydrogenase activity (soluble marker) in cell lysate. More importantly, the purified outer membranes contained less than 4% contamination by the C subunit of F1/F0 ATPase (inner membrane marker). Very little flagellin protein was present in the outer membrane sample. This indicated that the outer membranes were relatively free of contamination by cytoplasmic, inner membrane or flagellar components. The outer membrane fractions (rho = 1.19 g/cm3) contained 0.15 mg (dry weight) of protein per mg. Inner membrane samples (rho = 1.12 g/cm3) contained 0.60 mg (dry weight) of protein per mg. Freeze-fracture electron microscopy revealed that the outer membrane vesicles contained about 1,700 intramembranous particles per micron 2 while inner membrane densities for inner and outer membranes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and nonequilibrium pH gel electrophoresis-SDS-PAGE analyses of inner and outer membrane samples revealed several proteins unique to the inner membrane and 20 proteins that localized specifically to the outer membrane. This analysis clearly shows that the inner and outer membranes isolated by this technique are unique structures.     

Purification of Potato Leaf Plasma Membrane Protein pp34, a Protein Phosphorylated in Response to Oligogalacturonide Signals for Defense and Development

A potato (Solanum tuberosum L.) plasma membrane protein called pp34, the only known example of a plasma membrane protein that is phosphorylated specifically in response to defined Oligogalacturonide signals in plants, has been purified to apparent homogeneity. Polyclonal antibodies raised in rabbits against the purified pp34 protein immunoprecipitated a single thiophosphorylated protein species from potato plasma membranes, as analyzed by two-dimensional denaturing electrophoresis and fluorography. The pp34 antibodies also recognized a single protein in tomato (Lycopersicon esculentum L.) membranes that is thiophosphorylated in response to Oligogalacturonide elicitors, as demonstrated by western blotting and specific immunoprecipitation. These experiments confirm the identity of the tomato membrane protein as a pp34 homolog and establish the high monospecificity of the pp34 antibodies. This will permit further investigation of the role of protein phosphorylation in oligouronide signaling for defensive genes in potato and tomato plants.