Enzyme histochemistry of the choroid plexus in rat and squirrel monkey

Summary The reactions given for various oxidative and hydrolytic enzymes by the choroid plexus of the squirrel monkey and the rat brain have been studied in detail. The lining cells show strong activity for citric acid cycle and glycolytic pathways enzymes. The stroma shows strong activity for adenosine triphosphatase, alkaline phosphatase, adenosine monophosphatase and glucose-6-phosphatase. The peripheral part or luminal borders of the cytoplasm of the choroidal cells show strong activity for alkaline phosphatase, adenosine monophosphatase and adenosine triphosphatase, and a well developed thiamine pyrophosphatase positive Golgi complex, indicating their participation in the formation and transport of secretory material. The nucleoli of the lining cells give a positive reaction for glucose-6-phosphatase and adenosine triphosphatase. Acid phosphatase like the thiamine pyrophosphatase positive Golgi material is found all over the cytoplasm. The functional significance of these findings is briefly discussed.This work has been carried out with the aid of Grant No. FR-00165 from the Animal Resources Branch, National Institutes of Health and NASA Grant NGR-11-001-016. T. R. Shanthaveerappa in previous publications.     

Enzymes of carbohydrate metabolism in the developing endosperm of maize

A number of enzymes presumably implicated in starch synthesis were assayed at various stages of endosperm development ranging from 8 days to 28 days after pollination. Activity for invertase, hexokinase, the glucose phosphate isomerases, the phosphoglucomutases, phosphorylase I, uridine diphosphate glucose pyrophosphorylase, and the starch granule-bound nucleoside diphosphate glucose-starch glucosyltransferase was present at the earliest stage of development (8 days) studied. Activity was detectable for phosphorylase III, the soluble adenosine diphosphate glucose-starch glucosyltransferase, adenosine diphosphate glucose pyrophosphorylase, and sucrose-uridine diphosphate glucosyltransferase at 12 days. For phosphorylase II and cytidine diphosphate glucose pyrophosphorylase, activity was first detectable at the 14- and 16-day stages, respectively. Rapid increases in starch content are observed prior to detectable activity for adenosine diphosphate glucose pyrophosphorylase, the soluble adenosine diphosphate glucose-starch glucosyltransferase and phosphorylases II and III. For all enzymes, except invertase, activity per endosperm rises to a peak at 22 or 28 days. Greatest activity for invertase is found at 12 days with a steady decline thereafter. The pattern of invertase activity in comparison with that of sucrose-uridine diphosphate glucosyltransferase supports previous suggestions, that the latter plays a key role in the conversion of sucrose to starch. In addition to phosphorylases I, II, and III, multiple forms of glucosephosphate isomerase and phosphoglucomutase were detected.     

Studies on vinblastine-induced autophagocytosis in mouse liver. IV. Origin of membranes

The origin of the limiting membranes of autophagic vacuoles (AV) in mouse hepatocytes was studied by cytochemical techniques. Autophagocytosis was induced by an intraperitoneal injection of vinblastine (50 mg/kg). The marker enzymes used were adenosine triphosphatase for the plasma membrane, glucose-6-phosphatase for the endoplasmic reticulum and thiamine pyrophosphatase for the Golgi apparatus and the endoplasmic reticulum. All the three enzymes showed a characteristic localization in both control and vinblastine-treated hepatocytes. The space between the limiting membranes of a few apparently newly formed AV's showed weak glucose-6-phosphatase activity. Neither adenosine triphosphatase nor thiamine pyrophosphatase activities were observed on or between the AV membranes. It was suggested that endoplasmic reticulum membranes may be used as a source of AV membranes in hepatocytes. The lack of glucose-6-phosphatase activity in the limiting membranes even of most of the newly formed AV's suggests a transformation process of the membranes destined to form AV, during which the enzyme activity characteristic for endoplasmic reticulum may disappear from them.     

Comparative histochemical localization of some hydrolytic enzymes in mammalian epididymides

The activity of enzymes hydrolyzing sodium-beta-glycerophosphate, adenosine triphosphate, adenosine monophosphate, and glucose-6-phosphate in the epididymides of adult ram, rabbit, rat and hamster has been investigated histochemically. The enzymes hydrolyzing sodium-beta-glycerophosphate, adenosine triphosphate and adenosine monophosphate were rather similar in their distribution in the intertubular connective tissue of the epididymides of the four species studied. However, the distribution and activity of these enzymes in the stereocilia varied. There was a high activity in the stereocilia of the middle segments of ram, rabbit and rat and in the proximal part of the terminal segment of hamster epididymides. Glucose-6-phosphatase was distributed homogeneously in the epithelial cells of the epididymides throughout the duct. The possible functions of these enzymes in the mammalian epididymis are briefly discussed.     

Changes in the chemical composition and the enzymic activities of hepatic microsomes of the chick embryo during development

1. The values of the protein, RNA and phospholipid concentrations within the total microsomal fractions obtained from different stages of embryonic chick liver are compared. 2. Only the phospholipid content increases significantly with increasing developmental age. 3. The lack of membranes in the early stages of development and the relative constancy of RNA values during development suggests that some of the protein present at the early developmental stages is of a non-membranous non-ribosomal nature. 4. Glucose 6-phosphatase, adenosine triphosphatase, NADH(2)-cytochrome c reductase and diaphorase all increased in activity as development progressed. 5. Comparisons of submicrosomal fractions with respect to their protein, RNA and phospholipid content showed that in all embryonic stages fraction II (rough-membrane fraction) contained more than 60% of the proteins, RNA and phospholipid of the microsomal fraction. 6. Glucose 6-phosphatase was shown to be present predominantly in fraction II, whereas adenosine triphosphatase was present predominantly in fraction Iab (smooth-membrane fraction). 7. The significance of the differences between the smooth- and rough-microsomal fractions is discussed.     

Biochemical compartmentation of fish tissues. Distribution of gluconeogenic enzymes in the brain

1. Specific glucose-6-phosphatase and fructose-1,6-diphosphatase activity were found to be biochemically compartmentalized in four parts of the brain in nine nutritionally important fishes. 2. Glucose-6-phosphatase and fructose-1,6-diphosphatase activity were highest in the cerebrum and lowest in the cerebellum. 3. Piscivorous fishes had the highest gluconeogenic enzyme content, followed by catfishes and major carps. 4. After the liver and muscles, the various parts of the brain play an important role in carbohydrate metabolism. 5. A direct relationship between the stage of evolution and elevation of gluconeogenic enzyme levels was observed. 6. It is evident from the results and the discussion that evolution modifies the biochemical organization of fishes in general and of their brain in particular.     

Histochemistry of the chick esophagus and trachea. II. Enzymes, nucleic acids, proteins, carbohydrates, and fats

Tissues of White Leghorn embryos of stages 17–45 and chicks of one day, two days, and three weeks of age were frozen, sectioned in a cryostat and, where appropriate, were fixed in cold calcium formol. Acid phosphatase, non-specific esterase, adenosine triphosphatase, 5-nucleotidase, non-specific glycerophosphatase, nucleotidediphosphatase, and glucose-6-phosphatase were localized in these tissues. Ribonucleic acid, acid mucopolysaccharides, triglycerides, and neutral fats were localized in tissues fixed with FAA and embedded in paraffin. Positive acid phosphatase reactions were obtained in the epithelium of the trachea and esophagus at all stages of development. 5-nucleotidase was found in the muscularis mucosae of the esophagus at all stages. Non-specific esterase appeared with histodifferentiation of the esophageal epithelium. Ribonucleic acid was localized in the basal regions of the epithelium. Mucous glands of the esophagus are rich in ribonucleic acid and acid phosphatase at all stages of development. With histodifferentiation and the onset of secretion of sulfated acid mucopolysaccharides, the glands and their ducts become highly reactive for adenosine triphosphatase and nucleotide-diphosphatase, indicating a role of these enzymes in secretion.     

Studies on the rabbit appendix. I. Lymphocyte-epithelial relations and the transport of bacteria from lumen to lymphoid nodule

The proventriculus of White Leghorn chick embryos (stages 29–45) newly-hatched chicks, and adult chickens were frozen, sectioned in a cryostat and treated histochemically to identify localizations of alkaline and acid phosphatase, adenosine triphosphatase, 5-nucleotidase, nucleotide-diphosphatase, non-specific glycerophosphatase, glucose-6-phosphatase, non-specific esterase and succinic dehydrogenase. Ribonucleic acid, proteins and acid mucopolysaccharides were identified in tissues fixed in FAA. Acid phosphatase, nucleotide-diphosphatase, adenosine triphosphatase, succinic dehydrogenase, ribonucleic acid and proteins were present in the cells of the deep glands at all stages of development. Alkaline phosphatase and 5-nucleotidase were found only in mesenchymal derivatives of the proventriculus. After the chick begins swallowing and digesting albumen, enzymatic activity increased and non-specific esterase became very reactive. The surface epithelium is covered with a mucous coat. Ribonucleic acid, non-specific esterase, acid phosphatase and nucleotide-diphosphatase were localized in the basal portions of the epithelial cells. The functional significance of these different patterns is discussed.     

Studies on vinblastine-induced autophagocytosis in mouse liver

Summary The origin of the limiting membranes of autophagic vacuoles (AV) in mouse hepatocytes was studied by cytochemical techniques. Autophagocytosis was induced by an intraperitoneal injection of vinblastine (50 mg/kg). The marker enzymes used were adenosine triphosphatase for the plasma membrane, glucose-6-phosphatase for the endoplasmic reticulum and thiamine pyrophosphatase for the Golgi apparatus and the endoplasmic reticulum. All the three enzymes showed a characteristic localization in both control and vinblastine-treated hepatocytes. The space between the limiting membranes of a few apparently newly formed AV's showed weak glucose-6-phosphatase activity. Neither adenosine triphosphatase nor thiamine pyrophosphatase activities were observed on or between the AV membranes. It was suggested that endoplasmic reticulum membranes may be used as a source of AV membranes in hepatocytes. The lack of glucose-6-phosphatase activity in the limiting membranes even of most of the newly formed AV's suggests a transformation process of the membranes destined to form AV, during which the enzyme activity characteristic for endoplasmic reticulum may disappear from them.     

Feeding habits of the cuttlefish <Emphasis Type="Italic">Sepia</Emphasis> <Emphasis Type="Italic">officinalis</Emphasis> during its life cycle in the Sado estuary (Portugal)

The importance of cephalopods in marine ecosystems is well recognized as they serve as important predators and prey in many food chains. During a 1-year period, cuttlefish were monthly sampled in five areas throughout the Sado estuary, and the stomachs of 445 individuals were examined. Significant changes in the diet according to their life stage were revealed, but similar food habits were presented by males and females. Juvenile individuals (the smaller ones with no visible distinct gonads) preyed mainly upon small crustaceans, while the immature ones (maturity stages I and II) had the most diverse diet with brachyurans as the major prey group. The mature individuals (maturity stages III and IV) showed a clear preference for larger crabs and fishes. Although the cuttlefish are known for their opportunistic feeding behaviour, a preference for some prey according to length and behaviour could be perceived during this study. Seasonal differences in the diet were mostly due to the variation of individuals according to their maturity stage during the year.     

Enzymes that hydrolyze adenine nucleotides in platelets from breast cancer patients

The activities of NTPDase (EC 3.6.1.5, apyrase, CD39) and 5'-nucleotidase (EC 3.1.3.5, CD73) enzymes were analyzed in platelets from breast cancer patients. Initially, patients were compared in terms of length (years) of tamoxifen use. The following groups were studied: breast cancer patients who did not use tamoxifen, patients using tamoxifen for 1-48 months, patients using tamoxifen for 49-84 months, and controls (healthy subjects). Results demonstrated that adenosine triphosphate (ATP) hydrolysis was enhanced (F(3,114)=8.53; P<0.001) and adenosine diphosphate (ADP) hydrolysis was reduced (F(3,106)=5.09, P=0.002) as a function of tamoxifen use, while adenosine monophosphate (AMP) hydrolysis was unchanged. Next, patients were compared statistically according to disease stage, determined by the tumor-node-metastasis (TNM) staging system for classifying breast tumor. ATP hydrolysis was significantly elevated in patients with stage I and II breast cancer (F(4,113)=4.35; P=0.003), but was normal in patients with stage III and IV cancer. ADP hydrolysis was reduced in stages II to IV (F(4,105)=3.88, P=0.006) and AMP hydrolysis was elevated in stage II (F(4,105)=3.45 P=0.01), but was normal in stages III and IV. Platelet aggregation time was similar in all patients regardless of tamoxifen use or disease stage. Prothrombin time (PT) and activated partial thromboplastin time (APTT) were also within the normal range and similar among all groups. Similarly, fibrinogen and fibrin degradation product (FDP) were unchanged in all groups. In conclusion, our study demonstrated for the first time that hydrolysis of adenine nucleotides is modified in platelets from breast cancer patients taking tamoxifen.     

Adenosine triphosphatase activity of mycoplasma membranes

Rottem, Shlomo (Hebrew University, Jerusalem, Israel), and Shmuel Razin. Adenosine triphosphatase activity of mycoplasma membranes. J. Bacteriol. 92:714-722. 1966.-Adenosine triphosphatase activity of Mycoplasma laidlawii, M. gallisepticum, and Mycoplasma sp. strain 14 was confined to the cell membrane. The enzymatic activity was dependent on magnesium, but was not activated by sodium and potassium. Ouabain did not inhibit the adenosine triphosphatase activity of the mycoplasmas, and did not interfere with the active accumulation of potassium by M. laidlawii cells. Sulfhydryl-blocking reagents and fluoride inhibited the enzymatic activity, whereas 2,4-dinitrophenol was without any effect. Membranes of M. laidlawii hydrolyzed other nucleotide triphosphates and adenosine diphosphate (ADP), but at a lower rate than adenosine triphosphate (ATP). Nucleoside-2'-(3')-phosphates, ribose-5-phosphate, glucose-6-phosphate, and pyrophosphate were not hydrolyzed by the membrane preparations. It seems that the enzyme(s) involved in ATP hydrolysis by M. laidlawii membranes is strongly bound to the membrane subunits, which would account for the failure to purify the enzyme by protein fractionation techniques. The adenosine triphosphatase activity of mycoplasma membranes resembles in its properties that of similar enzymes studied in bacteria. The mycoplasma enzyme(s) seems to differ from the adenosine triphosphatase associated with ion transport in mammalian cell membranes and from mitochondrial adenosine triphosphatase.     

Calcium regulates Gladiolus flower senescence by influencing antioxidative enzymes activity

The objective of the present investigation was to study the role of calcium on antioxidative enzymes activity during the post-harvest life of Gladiolus (Gladiolus grandiflorus). Among the various calcium (Ca) treatments, 50 mmol l⁻¹ Ca treatments caused the highest increase in the vase life of the spike, from 5.5 days in control to about 9 days. Relative water content and membrane stability index (MSI) decreased from I to V stage. However, significant increase in relative water content and MSI were observed by 50 mmol l⁻¹ Ca as compared to control. Indices of oxidative stress such as lipid peroxidation and lipoxygenase activity increased from I to V stage, but decreased significantly in 50 mmol l⁻¹ Ca treatment. The activities of antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT) decreased initially from stage I to II, followed by an increase in stage III and thereafter started to decline at stages IV and V. Ascorbate peroxidase (APX) activity increased initially from stage I to III and thereafter declined in stage IV and V in both control and treatment. However, Ca with concentration of 50 mmol l⁻¹ increased the activities of SOD, CAT and APX at all the stages. The results revealed that spikes treated with Ca (50 mmol l⁻¹) solution maintained higher level of antioxidant enzymes activity and also showed delayed senescence in comparison to control.     

Three deoxyribonucleic acid-dependent adenosine triphosphatases from Bacillus subtilis.

We have isolated from Bacillus subtilis three deoxyribonucleic acid (DNA)-dependent adenosine triphosphatases (ATPases) (gamma-phosphohydrolases). The enzymes were extensively purified, and their physicochemical and functional properties were determined. The three enzymes (ATPases I, II, and III) were shown to be different by several criteria. ATPases II and III showed an absolute requirement for single-stranded DNA as a cofactor, whereas ATPase I had some residual activity also with double-stranded DNA. They required Mg2+ and had a pH optimum of 6.5 to 7. Only adenosine 5'-triphosphate and deoxyadenosine 5'-triphosphate were hydrolyzed. The molecular weights of ATPases I, II, and III were 108,000, 115,000, and 148,000, respectively. Km values for adenosine 5'-triphosphate and DNA were also evaluated and shown to be different for each enzyme. All three enzymes formed physical complexes with single-stranded DNA. We present evidence that ATPases I and II might migrate along DNA during adenosine 5'-triphosphate hydrolysis. On the other hand, this effect was not observed with ATPase III, which exhibited the highest affinity for single-stranded DNA.     

Carboxypeptidases of germinating triticale grains

Presence of five carboxypeptidases was found in endosperm of germinating triticale grains, while two of them in scutellum. Changes of their activities during four days of germination suggest that carboxypeptidase II plays an important role at initial stage of germination, while carboxypeptidases I and III - at subsequent stages of the process. High activity of carboxypeptidase II both in scutellum and endosperm of dry grains accompanied by its decrease during germination, and on the other hand, the appearance of carboxypeptidases I and III activities at the 2^nd and 3^rd day of the process seems to confirm such functions of these enzymes. Experiments with GA₃ indicated that carboxypeptidase I was synthesized in scutellum, and carboxypeptidase III — in aleurone layer. Carboxypeptidases I and II cleave N-CBZ-Phe-Ala, and carboxypeptidase III — N-CBZ-Ala-Met and N-CBZ-Ala-Phe as substrates with the highest rate.     

Studies on the histochemistry of phosphatase enzymes in the juxtaglomerular complex

Summary The localisation of alkaline-, adenosine tri-, glucose-6- and acid phosphatase was studied in the juxtaglomerular complexes of rat, mouse and human kidneys. An alkaline-and adenosine triphosphatase active region was observed between the macula densa, Goormaghtigh cell group and in the interstitium of the latter. The adenosine triphosphatase activity extended into the lateral cell membranes of the macular cells and in properly incubated sections it did not appear among other distal tubular cells. The granular juxtaglomerular cells were ATP-ase negative. The cells of the human macula densa and the granular juxtaglomerular cells of the rat and mouse showed acid phosphatase activity. The glucose-6-phosphatase reaction, accomplished at acid and alkaline pH, was negative in the JG complex of all three species. The possible role of these enzymes in the function of the JG complex also has been discussed.     

Changes in the stages of nocturnal sleep in man with a lesion of the hypothalamus-brain stem structures]

Polygraphic study of nocturnal sleep in 18 patients with affection of the hypothalamo-mesencephalic structures showed a reduction of the duration of the II stage and a prolongation of the III and IV stages of slow sleep in comparison with the corresponding indices in the patients with involvement of the ponto-bulbar structures (9) and in healthy persons (8) of control group. Analysis of the qualitative indices of the EEG of slow sleep (a relative amount of the sleep spindles per 1 min of the II stage, delta-index and the delta-wave amplitude at the II and the IV stages) demonstrated a tendency to their increase in the patients with affection of the hypothalamo-mesencephalic structures. Patients of this group showed a decreased frequency of spontaneous changes of sleep from deeper to more superficial stages.     

Phosphatases in the accessory respiratory organs of two fresh-water fishes.

The distribution of enzymes, viz., alkaline phosphatase, acid phosphatase, adenosine monophosphatase and adenosine triphosphatase was studied by histochemical methods in the accessory respiratory organs of two fresh-water fishes (Clarius batrachus and Heteropneustes fossilis). Enzymes have been used as markers to differentiate between functional and non-functional cells of the dendritic organ of Clarius and of the air chamber of Heteropneustes. The variations in the enzyme activities have been correlated with the functional capacity of each respiratory organ. It is attempted to understand the physiological role of these enzymes in the process of aerial breathing.     

Early stages of hepatic carcinogenesis after vagotomy

Initial stages of hepatocarcinogenesis have been studied in nonoperated and vagotomized animals. As a carcinogenic substance diethylnitrosamine (DENA) has been used. In order to estimate manifestation of the changes, the histochemical method for revealing glucoso-6-phosphatase activity, adenosine triphosphatase and gamma-glutamyltranspeptidase in the liver has been applied. The disturbance of vagus innervation is stated to delay the course of early stages of hepatocarcinogenesis, induced with DENA.