Histochemical and biochemical studies on esterase activity in the rat ovary

A correlative histochemical and biochemical study has been made of the changes in esterase positive sites in immature (10-, 20- and 30-days old), mature normal cycling (3-, 5- and 8-months-old), pregnant and lactating rat ovaries. The typical perivascular esterase-positive sites localized in the hilar portion, branch along the blood vessels and traverse into medullary and cortical portions of the ovary. The stromal vascularity surrounding the normal developing follicles, corpora lutea, atretic follicles and interstitial gland tissue showed rich activity of this enzyme system. On semiquantitative basis the number, intensity and quantity of esterase-positive sites vary with the maturation and reproductive states of the rat. The administration of estradiol-17 beta increased the fine perifollicular and theca externa perivascular esterase-positive sites, whereas atropine and reserpine affected severely both the large and fine meshwork of esterase-positive sites. Biochemical estimates of acetylcholine esterase activity endorse these histochemical observations. The possible roles of AChE activity in varied ovarian functions are discussed.     

Studies on the ultrastructural localization of adenosine triphosphatase activity in fracture callus

Summary The fine structural localization of Mg⁺⁺-activated and Mg⁺⁺-independent neutral adenosine triphosphatase (ATPase) was studied in fracture callus of the rat using EDTA-decalcified DMSO-treated tissues incubated in Wachstein-Meisel type lead-containing media, and N-ethylmaleimide, NaF, EDTA and histidine as inhibitors to test the specificity of the reaction. Final product was found to be deposited on the plasma membranes and associated structures (subplasmalemmal vesicles and vacuoles) of phagocytic monocytoid cells, fibroblasts, osteoblasts and ruffled border regions of osteoclasts when Mg⁺⁺ was present in the incubation medium; the most abundant precipitate was noted on the plasma membranes of osteoblasts. When Mg⁺⁺ was omitted from the medium, the ruffled borders of osteoclasts were the only plasmalemmal sites showing conspicuous activity. This apparently Mg⁺⁺-independent ATPase was also demonstrated in the lysosomes of all the different cell types in the callus and in the vacuoles and specific granules located beneath the ruffled border of osteoclasts; lack of inhibition with NaF suggested that the enzyme was not a conventional nonspecific acid phosphatase. Neither the Mg⁺⁺-activated nor the Mg⁺⁺-resistant ATPase were inhibited by EDTA or histidine, indicating that they were unrelated to non-specific alkaline phosphatase. Deposition of final product did not occur on the plasma membranes of chondroblasts, chondrocytes of osteocytes.     

Histochemical studies on the localization and activity of acid phosphatase in calcifying tissues

Summary Acid phosphatase activity has been studied in cold microtome sections and using simultaneous azo coupling method in developing teeth and bone, and serial sections were made for the demonstrations of alkaline phosphatase.1. In developing teeth, strongest activity of acid phosphatase was found in the distal portion of high columnar ameloblasts associated with heavy calcification in the rodent incisor, and ameloblasts and odontoblasts in adjacent occlusal surface in molar teeth. However, the activity of immatured ameloblast and crevicular aspects of molar were weaker.2. In the epiphyseal bone trabeculae a striking acid phosphatase reaction was found.3. As regards to the effects of decalcifying solutions to the enzymatic activity, the use of EDTA decalcifying agent (10% and pH 7 to 4) showed the best results. That is, a decrease of decalcifying time and a greater preservation of acid phosphatase activity.With 11 Figures in the Text     

Histochemical studies on the localization of oxidative and dephosphorylating enzymes and esterases in the peritoneal mesothelial cells

Summary Histochemical localization of various groups of oxidative and dephosphorylating enzymes and esterases has been made in the peritoneal mesothelial cells. These cells were strongly positive for lactic dehydrogenase and succinic dehydrogenase, moderately positive for cytochrome oxidase, simple esterase, acid phosphatase, 5nucleotidase and nonspecific cholinesterase, and negative for monoamine oxidase and specific cholinesterase. They showed moderate positive activity for alkaline phosphatase, localized in the microvilli of these cells. They contain scanty amount of thiamine pyrophosphatase-positive Golgi complex and showed diffuse thiamine pyrophosphatase-positive activity in the cell cytoplasm. Adenosine triphosphatase-positive spaces found in between the adjacent cells and at the bases of the individual cells have been demonstrated. These results are discussed in view of the great absorptive power of the peritoneal mesothelium.     

Histochemical and biochemical studies on the red and white muscle in rabbit

1. The total amount of triglyceride was 6.00 +/- 0.14 mg/g wet tissue in soleus, 1.50 +/- 0.52 in extensor digitorum longus and 1.83 +/- 0.88 in gastrocnemius muscle. 2. The amounts of triglycerides in the individual types were calculated to be very large, moderate and very small in type 1, 2A and 2B, respectively, when compared with histochemical studies. 3. Differences in fatty acid composition of triglycerides were seen between the soleus and extensor digitorum longus, and gastrocnemius showed intermediate values. 4. These results might be important corresponding to differences in energy metabolism in different fiber types.     

Ultrastructural localization of lysosomal enzymes in the egg cortex of Brachydanio

The localization of acid phosphatase (E.C. 3.1.3.2), inorganic trimetaphosphatase (E.C. 3.6.1.2), and aryl sulfatase (E.C. 3.1.6.1) in the cortex of unactivated and activated eggs of Brachydanio was examined by ultrastructural cytochemistry. Using a lead capture method, activity for all three acid hydrolases was demonstrated in organelles of the cortex before and after egg activation. Acid phosphatase (AcPase) reaction product was consistently present in primary lysosomes, secondary lysosomes, multivesicular bodies, and yolk bodies. AcPase activity was absent from mitochondria, profiles of the endoplasmic reticulum, coated pits of exocytosed cortical granules, and coated vesicles. Although most cortical granules of the mature, unactivated egg were unreactive for this enzyme, a few showed AcPase reaction product. It is not clear whether the AcPase-positive granules might be an immature form of cortical granules or a subpopulation of these organelles with lysosomal properties. Most cisternae of the Golgi apparatus did not stain for AcPase; however, reaction product was occasionally localized in a single cisterna as well as several small vesicles at the inner face of the Golgi. The intensity of the reaction product and the pattern of distribution of trimetaphosphatase (Tm-Pase) activity was very similar to that of AcPase. However, TmPase was never observed in cortical granules. Cortices of unactivated and activated eggs showed less overall aryl sulfatase (ArSase) activity when compared with AcPase and TmPase. The presence of ArSase reaction product in lysosomes and multivesicular bodies confirmed the acid hydrolytic nature of these organelles. AcPase and TmPase, and to a lesser extent ArSase, are adequate markers of a cortical lysosomal system in the danio egg.(ABSTRACT TRUNCATED AT 250 WORDS)     

Histochemical studies on the enzymes of glycogen metabolism in hamster epididymis

Summary Active and total (active + inactive) phosphorylase and glycogen synthetase (I- and D-form) were studied in hamster epididymis in relation to glycogen. Immature and adult, sexually active and regressed animals were examined.Epididymis in adult animals, based on their phosphorylase activity, may be divided into 5 zones. The zone 1 epithelium contains particulate glycogen, rich in phosphorylase and glycogen synthetase. The epithelial cytoplasm also contains moderate phosphorylase activity. The zone 2 epithelium is almost devoid of phosphorylase. The zone 3 epithelium shows considerable phosphorylase activity both in principal and holocrine cells. The epithelium of the zone 4 contains the highest total phosphorylase activity. In the zone 5 epithelium phosphorylase and glycogen are absent, but glycogen synthetase is often observed.Holocrine cells, particularly in zones 3 and 4, contain predominating active phosphorylase, some glycogen, but no synthetase activity. The lumen in the zone 4 often shows a faint staining for glycogen.In immature animals, low phosphorylase activity is always present in the epithelial cells. Holocrine cells are detectable, by their phosphorylase activity, in 4 week animals. The division of zones is usually established slightly before sexual maturation.During the period of sexual regression, phosphorylase diminishes considerably. Glycogen, phosphorylase and glycogen synthetase are, however, detectable in the zone 1 of these animals.     

Structural and biochemical studies of the distinct activity profiles of Rai1 enzymes

Recent studies showed that Rai1 and its homologs are a crucial component of the mRNA 5′-end capping quality control mechanism. They can possess RNA 5′-end pyrophosphohydrolase (PPH), decapping, and 5′-3′ exonuclease (toward 5′ monophosphate RNA) activities, which help to degrade mRNAs with incomplete 5′-end capping. A single active site in the enzyme supports these apparently distinct activities. However, each Rai1 protein studied so far has a unique set of activities, and the molecular basis for these differences are not known. Here, we have characterized the highly diverse activity profiles of Rai1 homologs from a collection of fungal organisms and identified a new activity for these enzymes, 5′-end triphosphonucleotide hydrolase (TPH) instead of PPH activity. Crystal structures of two of these enzymes bound to RNA oligonucleotides reveal differences in the RNA binding modes. Structure-based mutations of these enzymes, changing residues that contact the RNA but are poorly conserved, have substantial effects on their activity, providing a framework to begin to understand the molecular basis for the different activity profiles.     

Histochemical localization of oxidized glutathione-catalysing enzymes in human term placenta

Summary This paper reports a new cytochemical affinity technique for detecting oxidized-glutathione-binding enzymes by light microscopy and for carrying out fine structural analyses by means of oxidized glutathione labelled with colloidal gold. Albumin-colloidal gold particles were prepared. Oxidized glutathione, added to the solution, replaced albumin on the surface of the gold, thus forming a new histochemical reagent. In human placenta, oxidized-glutathione-catalysing activity was detected on the brush border of the placental villi, over the foldings of endothelial membranes, and in the ground substance of connective tissue in the villous core. Every process of the cell membrane demonstrated enhanced oxidized-glutathione-catalysing activity. This new reagent is a unique example of a low-molecular-weight compound labelled with colloidal gold, and it permits direct measurement of oxidized-glutathione-binding activity in tissue sections.