Quantitative histochemical assessment of the heterogeneity of glycogen phosphorylase activity in liver parenchyma of fasted rats using the semipermeable membrane technique and the PAS reaction

Summary Glycogen phosphorylase (EC 2.4.1.1) has been demonstrated in sections of liver from rats starved for 24 h. The method is based on the measurement of the amount of glycogen formed after incubation in a gelled medium containing glucose 1-phosphate as substrate, using the semipermeable membrane technique. Glycogen was demonstrated with the periodic acid-Schiff (PAS) reaction.Phosphorylase activity appeared to be highest in periportal areas. The optimum substrate concentration for revealing activity of the enzyme was 60–120mm. After incubation in the absence of substrate, the staining intensity, as measured cytophotometrically as the mean integrated absorbance at 560 nm, was similar to that of an unincubated section.p-Chloromercuribenzoate, a non-specific inhibitor of glycogen phosphorylase activity, reduced the formation of final reaction product attributable to phosphorylase activity completely. The Michaelis constants (K _m ) of the enzyme in periportal and pericentral areas differed. This was probably due to the presence of thea form only in periportal areas and of thea andb forms in pericentral areas. The mean integrated absorbances in both the periportal and pericentral areas increased linearly with incubation time (4–16 min). A linear relationship was also found with section thickness (4–10 µm). The total activity of glycogen phosphorylase in the periportal areas was double the pericentral activity.It is concluded that the semipermeable membrane technique, combined with the PAS reaction for glycogen, can be used as a valid method for the demonstration and quantification of glycogen phosphorylase activity in livers from starved rats.     

A new fixed cryosection technique for the simultaneous immunocytochemical demonstration of T6 and S100 antigens

Summary A formalin—calcium fixation method of preparing cryosections is described, which allows demonstration of Langerhans' cells by S100 antigen staining on frozen sections. The number of Langerhans' cells given by T6 antigen staining is also higher in formalin—calcium fixed frozen sections than acetone fixed frozen sections. The preparation is suitable for dual demonstration of the two antigens on the same section enabling a more accurate numerical evaluation of Langerhans' cell populations in the normal cervical epithelium.     

Arylsulphatase activity and cerebroside sulphates in the frog oviduct during the reproductive cycle

Summary The presence of arylsulphatase A and cerebroside sulphates in different tracts ofRana esculenta oviduct during different phases of the reproductive cycle were investigated by histochemical and biochemical procedures. The results indicate that enzyme activity shows seasonal fluctuations connected with the phase of the sexual cycle. The concentrations of cerebroside sulphates (the natural substrates of arylsulphatase A) is related to the activity of this hydrolytic enzyme. The role of arylsulphatase A activity in regulating the substrate concentration, and particularly that of sulphatides, is discussed.     

Biochemical and cytochemical comparison of intestinal bicarbonate-stimulated Mg2+ dependent ATPase and alkaline phosphatase activities in rat, rabbit and guinea pig

Summary Bicarbonate-stimulated Mg²⁺ dependent ATPase activity was demonstrated both biochemically and cytochemically, in brush border membranes from rat, rabbit and guinea pig duodenum. There was no correlation between enzyme activity and basal HCO ₃ ^- secretion rates in the different species. The concentration of HCO ₃ ^- necessary for optimal stimulation of ATPase activity, degree of stimulation and total activity was higher in the rat than in other species. Activity was higher in rat duodenum than in the ileum. This is consistent with the proposed electrogenic HCO ₃ ^- secretion in the duodenum. Distribution of activities of alkaline phosphatase and HCO ₃ ^- -stimulated Mg²⁺-ATPase along the duodenal villus showed significant differences, suggesting that the two activities reflect, at least in part, distinct enzymes.     

Chromosome Specificity of Polysomy Promotion by Disruptions of the Saccharomyces cerevisiae RNA1 Gene

Previously, we showed that a disruption of the yeast RNA1 gene with LEU2 sequences promotes polysomy for chromosome XIII. Here we demonstrate that this phenotype is due to sequences specific to the RNA1 gene and that the disruption allele does not affect nondisjunction of three other chromosomes or polysomy of a minichromosome. Hence polysomy appears to be restricted to chromosome XIII.     

A New Non-Mendelian Genetic Element of Yeast That Increases Cytopathology Produced by M1 Double-Stranded RNA in ski Strains

The Saccharomyces cerevisiae SKI (superkiller) genes are repressors of replication of M, L-A, and L-BC double-stranded (ds) RNAs; ski strains have an increased M dsRNA copy number and, as a result, are cold-sensitive for growth at 8 degrees. Growth is normal, however, at higher temperatures. We have found a new cytoplasmic genetic element [D] (for disease) that makes M1 dsRNA-containing superkiller strains grow slowly at 30 degrees, not at all at 37 degrees, and only very poorly at 20 degrees. These growth defects require three factors: a chromosomal ski mutation, the presence of M1 dsRNA, and the presence of the new cytoplasmic factor, [D]. We have isolated mutants unable to maintain [D] (mad), at least one of which is due to mutation of a single chromosomal locus. Further, [D] can be cured by growth at 37-39 degrees. We present evidence that [D] is not M, L-A, L-BC or W dsRNAs or mitochondrial DNA, 2 mu DNA, or [psi], but [D] depends on L-A for its maintenance. We also show that [D] is distinct from [B], a cytoplasmic element that allows M1 dsRNA to be stably replicated and maintained in spite of defects in certain chromosomal MAK genes that would otherwise be necessary. [D] activity is blocked by the presence of another extrachromosomal element, called [DIN] (for [D] interference). [D] and [DIN] may be different natural variants of the same molecule.     

Recalibration of the Pseudomonas aeruginosa Strain Pao Chromosome Map in Time Units Using High-Frequency-of-Recombination Donors

High-frequency-of-recombination donors of P. aeruginosa strain PAO were generated using a temperature-sensitive, replication mutant of the IncP-1 plasmid R68, loaded with the transposon Tn2521. Fourteen donors so isolated mobilized the chromosome in a polarized manner from a number of different transfer origins. The donors were used to construct a time of entry map of the entire chromosome and this was achieved by determining the time of entry of 32 randomly dispersed markers in crosses using nalidixic acid to interrupt chromosome transfer. Analysis of the time of entry data enabled the recalibration of the chromosome map to 75 min.     

Elimination of the non-specific binding of avidin to tissue sections

Summary A simple procedure is described for eliminating non-specific staining with avidin—peroxidase conjugates. Murine ovaries were embedded in either paraffin wax or epoxy resin and, after blocking endogenous peroxidase activity, were treated with 10 µg/ml biotinylatedPisum sativum agglutinin. Avidin—peroxidase conjugates (5 µg/ml), diluted in standard 0.05m tris-buffered saline, pH 7.6, containing 0.139m NaCl, produced considerable background coloration and intense mast cell staining in controls without the lectin. This background diminished as the ionic strength of the buffer was raised. At 0.125m Tris-buffered saline (containing 0.347m NaCl) the background was completely unstained, with elimination of all binding to mast cells and only minimal loss of specific lectin binding.     

The impact of different fixation procedures on staining of macromolecules in a microtiter system

Summary The effects of formaldehyde, glutaraldehyde, methanol, Clarke's fixative and microwave irradiation on the quantitative staining of proteins (Naphthol Yellow S) and nucleic acids (Ethyl Green—Pyronin) in a cell culture system have been investigated. Overall, glutaraldehyde rapidly yielded the highest and most consistent levels of staining when compared to all other chemical fixatives. Although microwave irradiation was found to be uneven, 4 min exposure to 700W was found to give higher levels of protein staining than those achieveable with glutaraldehyde. Time-dependent processes were observed with all procedures. In addition, dissociations in the trends of protein and nucleic acid staining were observed. It is suggested that these results domonstrate fixation events that have not previously been resolved from the effects of reagent penetration into tissue blocks.