Electron microscopic-cytochemical study of the enzyme activity of energy metabolism in "dark" and "light" neurons of the rat cerebral cortex

The ultrastructural localization of succinate dehydrogenase (SDH) and lactate dehydrogenase (LDH) activity in "dark" and "light" neurons of the intact rat's frontal brain cortex has been studied. The enzymes' activity was detected with using potassium ferricyanide as artificial acceptor of electrons. In the "light" cells SDH activity is localized in the mitochondria and plasma membranes. LDH activity is localized in the mitochondria, plasma membranes and hyaloplasm. SDH and LDH activity was not found in the "dark" cells.     

Differential response of the NADH oxidase of plasma membranes of rat liver and hepatoma and HeLa cells to thiol reagents

NADH oxidase activity of plasma membranes from rat hepatoma and HeLa cells responded to thiol reagents in a manner different from that of plasma membranes of liver. Specifically, the NADH oxidase activity of plasma membranes of HeLa cells was inhibited by submicromolar concentrations of the thiol reagentsp-chloromercuribenzoate (PCMB),N-ethylmaleimide (NEM), or 5,5-dithiobis-(2-nitrophenylbenzoic acid) (DTNB), whereas that of the rat liver plasma membranes was unaffected or stimulated over a wide range of concentrations extending into the millimolar range. With some hepatoma preparations, the NADH oxidase activity of hepatoma plasma membranes was stimulated rather than inhibited by PCMB, whereas with all preparations of hepatoma plasma membranes, NEM and DTNB stimulated the activity. In contrast, NADH oxidase activity of rat liver plasma membrane was largely unaffected over the same range of PCMB concentrations that either stimulated or inhibited with rat hepatoma or HeLa cell plasma membranes. Dithiothreitol and glutathione stimulated NADH oxidase activity of plasma membranes of rat liver and hepatoma but inhibited that of HeLa plasma membranes. The findings demonstrate a difference between the NADH oxidase activity of normal rat liver plasma membranes of rat hepatoma and HeLa cell plasma membranes in addition to the differential response to growth factors and hormones reported previously (Brunoet al., 1992). Results are consistent with a structural modification of a NADH oxidase activity involving thiol groups present in plasma membranes of rat hepatoma and HeLa cells but absent or inaccessible with plasma membranes of rat liver.     

Arrhenius plot characteristics of adipocyte-plasma-membrane adenylate cyclase activity in lean and genetically obese (ob/ob) mice

Arrhenius plots of fluoride- and guanine-nucleotide-stimulated adenylate cyclase activity were linear in adipocyte plasma membranes from lean and obese (ob/ob) mice . Arrhenius plots of isoprenaline-stimulated adenylate cyclase activity in hepatic plasma membranes biphasic in both groups. The results were biphasic in membranes from Jean mice but linear in membranes from obese mice. In contrast, Arrhenius plots of glucagon-stimulated adenylate cyclase activity in hepatic plasma membranes were biphasic in both groups. The results suggest that the coupling between the -receptor and the regulatory unit of adenylate cyclase, which has been observed to be defective in adipocyte plasma membranes from obese mice, is influenced by a different lipid environment in membranes from obese animals.     

PREPARATION AND CHARACTERIZATION OF A PLASMA MEMBRANE FRACTION FROM ISOLATED FAT CELLS

A rapid method of preparing plasma membranes from isolated fat cells is described. After homogenization of the cells, various fractions were isolated by differential centrifugation and linear gradients. Ficoll gradients were preferred because total preparation time was under 3 hr. The density of the plasma membranes was 1.14 in sucrose. The plasma membrane fraction was virtually uncontaminated by nuclei but contained 10% of the mitochondrial succinic dehydrogenase activity and 25–30% of the RNA and reduced nicotinamide adenine dinucleotide cytochrome c reductase activity of the microsomal fraction. Part of the RNA and NADH-cytochrome c reductase activity was believed to be native to the plasma membrane or to the attached endoplasmic reticulum membranes demonstrated by electron microscopy. The adenyl cyclase activity of the plasma membrane fraction was five times that of Rodbell's "ghost" preparation and retained sensitivity to epinephrine. The plasma membrane ATPase activity was five times that of the homogenate and microsomal fractions. Electron microscopic evidence suggested contamination of the plasma membrane fraction by other subcellular components to be less than the biochemical data indicated.     

Inhibition of the NADH oxidase activity of plasma membranes isolated from xenografts and cell lines by the antitumor sulfonylurea,N-(4-methylphenylsulfonyl)-N′-(4-chlorophenyl)urea (LY 181984) correlates with drug susceptibility of growth

Summary Inhibition of NADH oxidase activity of plasma membranes isolated from a series of human xenografts and cell lines by the antitumor sulfonylurea, N-(4-methylphenylsulfonyl)-N-(4-chlorophenyl) urea (LY 181984), correlated with the ability of the sulfonylurea to inhibit cell growth. Growth of rat kidney cells either untransformed or transformed with Kirsten-ras (K-ras) were unaffected by the sulfonylurea. Similarly, the NADH oxidase activity of isolated plasma membranes from K-ras transformed cells was unaffected by LY 181984. In contrast, when transformed with Harvey-ras (H-ras), both growth and NADH oxidase activity were inhibited. With the inactive but structurally related LY 181985 (N-4-methylphenyl-sulfonyl)-N-(phenyl)urea), neither growth nor plasma membrane NADH oxidase activity of either sulfonylurea-susceptible or -resistant tissues or cell lines was inhibited. Both sulfonylureas were inactive with rat liver plasma membranes but NADH oxidase activity of plasma membranes and growth with HeLa cells was inhibited by the active (LY 181984) but not by the inactive (LY 181985) sulfonylurea. The findings suggest a possible correlation between inhibition of plasma membrane NADH oxidase activity by the antitumor sulfonylureas and their oncolytic action.     

Ultracytochemical localizations of adenylate cyclase,guanylate cyclase and cyclic 3′,5′-nucleotide phosphodiesterase activity on the trophoblast in the human placenta

Summary Ultracytochemical localizations of cyclic nucleotide-metabolizing enzymes, namely adenylate cyclase (AC), guanylate cyclase (GC) and cyclic 3,5-nucleotide phosphodiesterase (PDE), have been demonstrated in the human term placenta. AC activity was found positive on the basal plasma membrane of the syncytiotrophoblast and on the pinocytotic vesicle of the fetal capillary endothelial cell. GC activity was observed to be strong on the plasma membrane of the microvilli of the syncytiotrophoblast. The cAMP PDE activity was shown positive both on the basal plasma membrane and on the microvillous membrane, while cGMP PDE activity was exclusively confined to the microvilli of the syncytiotrophoblast. These observations suggest that the syncytiotrophoblast plays an important role in the cyclic nucleotide metabolism in the human term placenta and that there might be significant functional differences between its basal plasma membrane and its microvillous membrane.     

Plasma trehalase activity and diabetes mellitus

Trehalase is an enzyme which hydrolyzes the disaccharide trehalose, yielding glucose. It is widespread in nature and found in various human tissues as well as in human plasma. The synthesis and degradation of its substrate trehalose have been considered as being implicated in carbohydrate transport mechanisms. Trehalase activity has been examined in both normal subjects and diabetic patients. In the normal subjects, the frequency histogram of the enzyme activity is bimodal, indicating the existence of genetic polymorphism. The proposed model of a single autosomal locus with two alleles has been verified, with 27% of the population tested belonging to the "low-activity" phenotype and 73% being of the "high-activity" phenotype. Males have higher mean plasma trehalase activity than females. Apparently, the reverse appears to be the case in the diabetic subjects. The mean value for all nondiabetics and that of diabetics were computed and the difference was found to be statistically significant (F = 7.02, N1 = 3, N2 = 56, P less than 0.01). An experiment showed that neither the abnormally high concentration of glucose in diabetics nor any other constituent of the diabetic plasma caused an increase in plasma trehalase activity (t = 0.0724, P greater than 0.10). A Woolf and Haldane test to determine association of diabetes mellitus and plasma trehalase phenotype indicated a highly significant association with the high-activity phenotype (chi 2 = 18.5350, P less than 0.01). Thus the inference is that people with high plasma trehalase activity are more prone to develop diabetes mellitus than people with low enzyme activity.     

A three hours study of the response of plasma corticosterone and of three liver enzymes in rats subjected to stress in the late afternoon or during the morning hours.

In adult male rats kept under controlled illumination (lights on from 6 a.m.--6 p.m.) the existence of higher afternoon values of plasma corticosterone ("B") is reconfirmed. Moreover, plasma "B" increases induced by 400 revolutions in Noble-Collip drums or immobilization for 2.5 h were found substantially less pronounced if stressor application took place at 4 p.m. rather than 8 a.m. so that essentially no difference between morning and afternoon poststress peaks was observed. Also the response to 0.05 mg ACTH i.p. was smaller in the afternoon experiments. Additionally three liver enzymes the activity of which is increased by corticosterone released under conditions of stress were also studied. Higher afternoon activity of liver tyrosine aminotransferase (TAT) and tryptophan pyrrolase (TP) was observed in spite of fasting from 8 a.m. imposed to the animals taking part in the afternoon experiments. The activity of phosphoenolpyruvate carboxykinase (PEPCK) was also increased in the afternoon probably because of the food deprivation. Immobilization-induced increase in the activity of all three enzymes showed a similar blunting as plasma "B" response, so that no differences between morning and afternoon were found in peak poststress values. In conclusion, the low plasma "B" response of rats at a time when the animals begin the active part of their day seems to have important metabolic implications.     

Melatonin and the circadian rhythms of feeding and perch-hopping in the European starling,Sturnus vulgaris

Summary The role of melatonin plasma titers in the control of free-running circadian rhythms was investigated in European starlings,Sturnus vulgaris, held in continuous dim light. Simultaneous recordings of plasma melatonin, perch-hopping and feeding activity revealed synchronous circadian variations in all three functions with high melatonin titers during resting and low titers during activity periods. Implanting birds with melatonin silastic capsules resulted in a 100-fold increase in plasma concentrations and an abolition of the endogenous melatonin rhythm. Feeding rhythms persisted during this treatment, although the activity time () was lengthened and the circadian period () shortened. Similar changes were found in perchhopping activity, but in some birds, this activity was more or less suppressed. These data demonstrate that although melatonin plasma titers freerun synchronously with other circadian parameters, fluctuations in peripheral melatonin are not essential for the expression of circadian activity.     

Disorder of electromechanical coupling in the cells of the myocardium in protracted crush syndrome

Experiments on papillary muscles of normal (control) rabbits and of those with the compression syndrome (CS) were made to explore the action of the control and "syndromic" blood plasma on electric and contractile activity of the myocardium. Isometric contractions of myocardial preparations were recorded at varying stimulation frequencies (0.1-2 Hz). Intracellular rest potentials (RP) and action potentials (AP) were led away with the aid of glass microelectrodes filled with 2.5 M KCl. The replacement of Tyrode solution by the control plasma raised the amplitude of papillary muscle contractions, that being greater as regards the muscles from rabbits with the CS. The "syndromic" plasma (diluted by Tyrode solution in a 1:1 ratio) markedly inhibited the amplitude of contractions of papillary muscles from both the control rabbits and animals with the CS. Reduction of the contractions induced by the "syndromic" plasma seen in all the preparations was followed by two patterns of changes in electrical activity of myocardial fibers. In one pattern, the RP, the amplitude and duration of the AP declined. In the other, on the contrary, the changes were reduced to a greater AP duration. The conclusion is made about the absence of a direct relationship between the decrease in myocardial contractility and changes in intracellular potentials induced by the "syndromic" plasma. It is suggested that the "syndromic" plasma deranges the process of stimulation and contraction coupling in heart papillary muscles.     

A possible mechanism for cholesteryl ester formation during demyelination

Lecithin: cholesterol acyltransferase (LCAT) activity has been examined in the rat by using a brain homogenate preparation as the phospholipid substrate and blood plasma as the enzyme source. LCAT activity was detected on using 60 l of serum onwards. Successive experiments have also shown that LCAT activity is present in the edematous rat brain tissue homogenate when incubated with inactivated rat plasma as substrate. The results are discussed in relation to cholesteryl ester accumulation in brain during demyelinating diseases.     

(1–3)-β-Glucan synthesis ofNeurospora crassa

(1–3)--d-Glucan synthase activity ofNeurospora crassa was localized to the plasma membrane by autoradiography of colloidal gold-labeled plasma membranes. The active site of glucan synthase for substrate hydrolysis was determined to be cytoplasmic facing. However, glucan synthase activity present in intact protoplasts was partially sensitive to Novozym 234 and to glutaraldehyde treatments, suggestive that enzyme activity is transmembrane. Enzyme activity also directed the formation of microfibrils in vitro. Taken together, these and previous results support the following scheme for glucan synthesis: 1. The sequential addition of glucose residues from UDP-glucose to glucan chains occurs on the cytoplasmically facing portion of glucan synthase. 2. As each glucan chain is synthesized, it is extruded to the extracytoplasmic side of the plasma membrane. 3. As each chain is extruded, it forms interchain hydrogen bonds with adjacent chains, resulting in glucan microfibril assembly.     

Evaluation of plasma muscle enzyme activity as an indicator of lesion characteristics and prognosis in horses undergoing celiotomy for acute gastrointestinal pain

Background

In horses undergoing celiotomy for acute gastrointestinal pain, identification of variables correlating with lesion severity and location, and survival provide veterinarians and owners with information that aids in making informed decisions regarding appropriate treatment. Muscle enzyme activity is often increased in horses undergoing celiotomy for acute gastrointestinal pain and it is not known if muscle enzyme activity increase is specific to lesion type or impacts prognosis for survival. The objective of this study was to evaluate the relationship of pre-operative increase in muscle enzyme activities with intestinal lesion characteristics, specifically lesion location (large versus small intestine) and whether it was strangulating versus nonstrangulating, and case survival in horses undergoing celiotomy for acute gastrointestinal pain.

Methods

Records of 241 horses undergoing exploratory laparotomy for colic were reviewed retrospectively. Evaluation of preoperative plasma aspartate aminotransferase (AST), creatine kinase (CK), sorbitol dehydrogenase (SDH), and gamma-glutamyltransferase (GGT) activities, fibrinogen and glucose concentrations, and hematocrit (HCT) and their association with gastrointestinal lesion characteristics and survival was performed.

Results

Pre-operative increase in plasma CK and AST activity, and HCT and decrease in plasma bilirubin concentration were significantly associated with presence of lesions resulting in intestinal ischemia. Increase in plasma CK activity and HCT were significantly associated with a decreased probability of survival to hospital discharge. Plasma GGT and SDH activity, and glucose and fibrinogen concentration were not significantly associated with survival or severity of disease in multivariate analysis.

Conclusions

Plasma muscle enyzme activity may be useful as a prognostic indicator in equine colic cases. Given that increases in plasma CK and AST activity were significantly associated with nonsurvival and the presence of intestinal ischemia, preoperative increase in these enzyme activities could assist in identification of disease severity and prognosis of horses undergoing celiotomy for acute gastrointestinal pain. Further study is indicated to elucidate the etiology of increased muscle enzyme activity in horses with surgical colic disease observed in this preliminary study.

     

Decrease of the plasma membrane H+-ATPase activity during late exponential growth of Saccharomyces cerevisiae

During the last cell division of exponential growth, the H+-ATPase activity from the yeast plasma membrane decreases by a factor of two to three. This "arrest growth control" of ATPase activity is not accompanied by modification of the sensitivity to vanadate.     

The inhibition of bovine ceruloplasmin oxidase activity by thiomolybdates in vivo and in vitro: a reversible interaction

The administration of pharmacological doses (greater than 100 mg Mo) of trithiomolybdate or tretrathiomolydbate to ruminants causes a transient apparent decrease in the ceruloplasmin oxidase activity of plasma and a more persistent increase in copper bound to plasma albumin. Sephadex gel-filtration and/or dilution of "inhibited" samples taken from an infused animal or of plasma treated with thiomolybdate in vitro restores activity back to pretreatment levels. The increase in albumin bound copper does not appear to be related to ceruloplasmin breakdown. It is concluded that, contrary to a recent report, the inhibition of ceruloplasmin oxidase activity is reversible and thus unlikely to be of pathological relevance, since circulatory thiomolybdate concentrations in molybdenotic animals are likely to be very low. It is recommended that thiomolybdate preparations used for in vitro and in vivo studies should be carefully purified by Sephadex chromatography.     

Localization of 3′, 5′-cyclic nucleotide phosphodiesterase activity in isolated thyroid cells and intact thyroid tissue

Summary Cytochemical localization of 3,5-cyclic nucleotide phosphodiesterase (cPDEase) has been investigated by light and electron microscopy in dissociated bovine thyroid cells and in intact bovine thyroid tissue. By light microscopy in isolated thyroid cells reaction product deposition associated with cPDEase activity was localized at the level of the plasma membrane. In intact cryostat cut thyroid tissue, the activity was primarily observed in the cytoplasm and to a lesser extent at the level of the plasma membrane. By electron microscopy, cPDEase activity in isolated cells was found on the plasma membrane and was also encountered on the inner surface of membrane bound vacuoles, presumably pinocytic in origin. In intact tissue, cPDEase activity appeared mostly localized on the apical and lateral plasma membranes and was also present on the outer surface of the endoplasmic reticulum (ER).Even though cPDEase and 5-AMPase did share the same plasma membrane localization, the inhibitory response to theophylline and stimulation with Imidazole permitted the dissociation of their respective activities. 5-AMPase failed to respond to either theophylline or Imidazole suggesting absence of cross reactivity between 5-AMP and cyclic AMP. Thyrotropin (TSH) had no effect on cPDEase activity.We conclude that: (1) regardless of the nature of the material used, the cytomembranes of thyroid cells possess cPDEase activity; and that (2) the variability in distribution as well as in staining intensity recorded by light and electron microscopy between isolated thyroid cells and cryostat cut thyroid tissue is probably inherent to the methodology used.This paper was presented, in part, at the 60th Annual Meeting of the International Academy of Pathology, Montreal, Canada, March 1971 and was initiated in the Department of Pathology, Rhode Island Hospital, Providence, R.I., supported from a Grant-in-Aid of the American Cancer Society, Rhode Island Division, Inc. and the Brown-Hazen Fund.     

Increased plasma α (1 → 3)-l-fucosyltransferase activities in patients with hepatocellular carcinoma

Alpha(1 3)-l-fucosyltransferase (1,3FT) activity was determined in plasma of patients with chronic liver diseases, namely, chronic hepatitis (CH), liver cirrhosis (LC) and hepatocellular carcinoma (HCC). The plasma 1,3FT activity was significantly higher (p<0.01) in chronic liver diseases than that in normal controls. The enzyme activity in plasma of patients with HCC was also significantly higher than that in LC (p<0.05) or that in CH (p<0.01). However, no significant difference was observed in the enzyme activity between LC and CH. Plasma 1,3FT activity in patients with HCC was not significantly changed before and after transcatheter arterial embolization. In addition, the enzyme activity in the homogenate of the cirrhotic liver tissue was higher than that in the preparation of the hepatoma tissue in the same patient. These results suggest that the increased plasma 1,3FT activity in patients with HCC reflects mainly the enzyme activity of cirrhotic liver tissue, not that of hepatoma tissue. The significance of the elevated levels of plasma 1,3FT and its decreased hepatoma tissue activity in patients with HCC, compared with that in LC, remains to be clarified.     

Increased vesicle endocytosis due to an increase in the plasma membrane phosphatidylserine concentration.

Endocytosis vesiculation consists of local membrane invaginations, continuously generated on the plasma membrane surface of living cells. This vesiculation process was found to be activated in vivo by the generation of a transmembrane surface area asymmetry in the plasma membrane bilayer, after enhancement of transbilayer phospholipid translocation. The observed enhancement was shown to be in good quantitative agreement with a theoretical model of elastic equilibrium describing stabilization of 100-nm vesicles in response to phospholipid redistribution. Very rapid dynamic vesiculation and direct re-fusion of the vesicles, both dependent on the phospholipid translocation activity, were found on a time scale of seconds. Both vesiculation and re-fusion were shown to result in a steady-state population of internal vesicles at long time points. The plasma membrane appears to be a dynamic structure, oscillating between two distinct curvature states, the 10 microns-1 "vesicle" and the 0.1 micron-1 "plasma membrane" curvature states. This dynamic behavior is discussed in terms of an elastic control of the membranes curvature state by the phospholipid translocation activity.     

Cytochemistry of gamma-glutamyltransferase in haemic cells and malignancies

Summary An improved cytochemical method for the demonstration of -glutamyltransferase is described. The enzyme was demonstrated in almost all normal leucocytes and in platelets. There was markedly reduced activity in most lymphoproliferative disorders. In a single case of Hodgkin's disease, T-lymphocytes showed slight to moderate activity contrasting with the marked activity displayed by Reed—Sternberg cells. The plasma cells of multiple myeloma and plasma cell leukaemia showed activity equal to or stronger than that of their normal counterparts. In acute myeloid leukaemia the positivity varied widely in blast cells, but was consistently increased in monoblasts of acute monoblastic leukaemia. -Glutamyltransferase may serve as a differentiation marker in the study of granulocytic and lymphocytic cell lineages.     

Aging-associated increase in indoleamine 2,3-dioxygenase (IDO) activity appears to be unrelated to the transcription of the IDO1 or IDO2 genes in peripheral blood mononuclear cells

Background

Old age is associated with increased levels of circulating pro-inflammatory cytokines, a phenomenon termed inflamm-aging. Elevated levels of pro-inflammatory cytokines have been associated with several age-associated diseases and with a shortened lifespan. Indoleamine 2,3-dioxygenase (IDO) has immunomodulatory properties and its activity is elevated in inflammation, autoimmune disorders and malignancies. We have previously shown that IDO activity is increased in nonagenarians compared to young individuals and that high IDO activity is associated with mortality at old age.

Findings

In this study our aim was to assess whether this difference in IDO activity in the plasma was due to the differential expression of either the IDO1 or IDO2 gene in peripheral blood mononuclear cells. Our results show that IDO1 and IDO2 are not differently expressed in nonagenarians compared to controls and that the expression of IDO genes is not associated with the level of IDO activity in the plasma.

Conclusion

The level of IDO activity in the plasma is not regulated through the expression of IDO1 or IDO2 in the peripheral blood mononuclear cells.