Studies on the molecular arrangement of RNA in tissues with a selective topo-optical reaction of RNA

Summary Selective, demonstration of RNA in tissues was achieved by treating tissue sections with potassium permanganate followed by bisulfite and toluidine blue at pH. 1.0 (PBT reaction). It is suggested that this reaction is due to aldehyde groups which are formed by the oxidative cleavage of the pyrimidine rings of RNA which can be selectively demonstrated using bisulfite-toluidine blue as the aldehyde reagent.The specificity of the reaction was tested after RNAase treatment, after acid hydrolysis, and on pure RNA droplets. The aldehyde nature of the reacting groups was checked, after permanganate oxidation, by Schiff's leucofuchsin reagent, and by aldehyde blocking reactions.Two types of intracellular molecular arrangement of RNA molecules could be distinguished by polarization optics after application of the PBT reaction: 1) The strong birefringence, dichroism and metachromatic staining of membrane-bound RNA in ergastoplasm of pancreas, liver and plasma cells indicate a linear (planar) molecular order of RNA molecules on the surface of the membranes, and 2) the isotropic, basophilic staining of RNA not organized in membrane structures (Nissl substance, nucleoli) suggest a random distribution of their dye binding sites.     

Demonstration of microorganisms in tissues by the ABT and KOH-ABT topo-optical reactions.

The aldehyde-bisulphite-toluidine blue (ABT) reaction, as a selective topo-optical test of vicinal OH and amino-OH groups is suited for the selective demonstration in tissues of microorganisms of polysaccharide containing cells. Alkaline pretreatment of the polysaccharide cell walls, releases, by splitting the O-acyl radicals, further vicinal OH groups for the ABT reaction, thus actually increases the sensitivity of the method. The topo-optical reactions are characterized by a strong birefringence induced by oriented dye-binding, due to the linear arrangement of polysaccharides composing the cell wall. Differences in the character of birefringence have made it possible to work out a new method for the analysis of the cell wall ultrastructure as well as to demonstrate microorganisms in tissues. The practical value of the reactions is illustrated by examples.     

Studies on the low molecular weight RNA associated with 28S ribosomal RNA from Crotalus durissus terrificus liver.

A low molecular weight RNA was released from the purified rattlesnake 28 S RNA by brief heat treatment as well as by treatment with 80% dimethylsulfoxide or formamide. The sedimentation coeficient of this low molecular weight RNA was found to be 5.5 S, corresponding to a nucleotide number of 140 and a molecular weight of 46 000. It was also observed that 5.5S RNA is present in equimolar ratio to 5 S rRNA. Heat treatment of the purified 60 S ribosomal subunit also released the 5.5 S RNA. The possibility that this low molecular weight RNA is located on the surface of the large ribosomal subunit is discussed.     

A note on the physiological arrangement of tissues

Presented are the kinetics of uptake of an inert solute by tissues arranged in series, distinct parallel and competitive parallel, with respect to the circulation. The uptake in all cases can be described by a series of exponentials, but the number of these terms and the character of the constants is specific for each arrangement. This fact can be of value in deducing tissue arrangement from uptake data. The material in this article is the opinion of the writers, and should not be construed as representing the policies of the Navy Department or the Naval Service at large.     

Studies on the asymmetric arrangement of membrane-lipid-enveloped virions as a model system.

Lipids of BHK 21 cells (baby hamster kidney) grown in tissue culture were labelled with radioactive fatty acids. The enveloped vesicular stomatitis virus was propagated in this host cell type. The virions were purified by density gradient centrifugation. Neuraminidase treatment of the intact virions led to a complete transformation of hematoside [N-acetylneuraminosyl(alpha2-3)lactosyl(beta1-1)ceramide] into lactosylceramide, with identical labelling of the ceramide portion in hematoside of the untreated virions and the lactosylceramide of the neuraminidase-treated particles. The morphology of the virions appeared unchanged in electron micrographs, but the neuraminic-acid-free virions had a strong tendency to aggregate. The results of these studies are evidence that gangliosides are integrated exclusively into the outer lamella of the lipid bilayer in the viral envelope. It is also evident that the viral envelope is a suitable model for studies on membrane asymmetry.     

A new rapid method for selective extraction of RNA from fixed mammalian tissues.

Treatment of formalin-fixed mammalian tissues with concentrated or 50% phosphoric acid at 5 degrees C for 20 and 50 min. respectively reveals complete extraction of RNA as judged by methyl green followed by staining with pyronin. This procedure also causes depolymerisation of DNA as indicated by the red staining of the nuclei. Sections treated with concentrated phosphoric acid at 5 degrees C for 30 min. causes disruption of the double helical structure of DNA what results in the depression of the pyronin staining. Similarly treated sections show Feulgen positive nuclei. Treatment of sections in 25 % phosphoric acid at 60 degrees C for 15 min. followed by staining with methyl green and pyronin show red nuclei, nucleoli and the cytoplasm. This indicates that extraction of RNA is only possible in cold and not at elevated temperature.     

Studies on the reaction mechanism of general acyl-CoA dehydrogenase. Determination of selective isotope effects in the dehydrogenation of butyryl-CoA

The kinetic properties of general acyl-CoA dehydrogenase from pig kidney have been investigated using normal butyryl-CoA as well as an alpha-deutero, beta-deutero- and perdeutero-butyryl-CoA. In turnover catalysis, isotope effects of 2, 3.6, and 9 were found respectively. In the reductive half reaction the isotope effects were 2.5, 14, and 28 for the same substrates, and 21 for (2R,3R)-(2,3-D2)butyryl-CoA. No intermediates are apparent during the reduction of oxidized enzyme to the presumed complex of reduced enzyme and crotonyl-CoA. The results are interpreted as indicating a high degree of concertedness during the rupture of the alpha and beta C-H bonds. They are compatible with a mechanism in which simultaneously the alpha-hydrogen is abstracted as a proton, while the beta-hydrogen is transferred to the oxidized flavin as a hydride.     

Studies on the selective chemical inhibition by urea of alkaline phosphatase isoenzymes in the reaction course

Urea inhibits the activity of alkaline phosphatase during the reaction course. The inactivation is progressively stronger for the placental, intestinal and renal subforms. Influence of reaction temperature, pH, type and molarity of buffer, magnesium chloride, albumin and enzyme concentration on the inactivation mechanism is evaluated. In all experimental conditions the process follows pseudofirst-order kinetics and the inactivation profiles are distinct and typical for each enzymatic subform. With a simple graphical analysis, a single inactivation curve in controlled experimental conditions, allows the identification of each isoenzyme from the slope and the calculation of the respective fractional amount from the intercept of the time-activity plot.     

Studies on the arrangement of nucleoprotein in elongate sperm heads

Sperm from Nucella lapillus and Gallus domesticus were treated with the enzymes pronase and trypsin and 8M urea to break down the nuclei. Preparations were dried by the critical point method and rotary shadowed with platinum/ carbon prior to examination with the electron microscope. The patterns of breakdown in the nuclei seen after experimental treatment show a close correlation with the patterns of nuclear condensation which occurs during spermiogenesis. No evidence was found to indicate boundaries between chromosomes. The arrangement of chromosomes within sperm nuclei and condensation patterns in spermiogenesis are discussed.Work supported in part by NSF University Science Development Award GU-1154.     

Caged RNA: photo-control of a ribozyme reaction.

We report here the first photo-chemical control of a ribozyme reaction by the site-specific modification of the 2'-hydroxyl nucleophile in the hammerhead system with a caging functionality. Rapid laser photolysis of the O-(2-nitrobenzyl) caging group initiates an efficient and accurate hammerhead-catalyzed cleavage of substrate RNA under native conditions. RNAs in which reactive functionalities or recognition elements are caged in this manner will be useful tools to probe RNA reactivity and dynamics.