Dexamethasone inhibition and phorbol myristate acetate stimulation of plasminogen activator in human embryonic lung cells

Treatment of human embryonic lung cells with dexamethasone resulted in a decrease in plasminogen activator activity measured in the fibrinolytic assay. The decrease in activity could at least partially be explained by the presence of an inhibitory substance(s) based on the following observations of lysates of dexamethasone-treated vs. control cells: a) an increase in specific activity following subcellular fractionation; b) an increase in fibrinolytic activity following separation by gel electrophoresis; c) an increase in fibrinolytic activity following mild acid-treatment; and d) a decrease in urokinase-directed fibrinolytic activity in mixing experiments. Phorbol myristate acetate increased plasminogen activator activity without affecting the level of inhibitory substance.     

Involvement of amygdala central nucleus in the regulation of cardiovascular functions

Effects of high frequency stimulation of the amygdala central nucleus involved a selective decrease in the activity: an increase in the activity of the inferior cardiac nerve and a simultaneous decrease in the activity of the vertebral nerve, as well as an obvious BP increase. Bilateral electrolytic lesions of the same amygdala structure accompanied with an overload of the higher nervous activity induced no hypertensions. The role of the amygdala central nucleus in control of cardiovascular functions is discussed.     

The effect of sex hormones on the acinar distribution pattern of phosphoenolpyruvate carboxykinase activity in rat liver

The intra-acinar distribution pattern of phosphoenolpyruvate carboxykinase activity (PEPCK) was investigated in microdissected samples of livers from normal, castrated, castrated and estradiol- or testosterone-treated, and uncastrated and testosterone- or estradiol-treated male and female rats. The total PEPCK activity showed a marked sex dependency, with 1.8 times higher activity in males. The intra-acinar distribution profiles were also sex-dependent. The periportal-to-perivenous gradient was steeper in males. Castration resulted in an approximation of PEPCK activity and its acinar distribution pattern between the sexes due to a reduction in males and an increase in females. Estrogen treatment of castrated males had no further effect on PEPCK activity and its acinar gradient, whereas in ovariectomized animals the activity was reduced to levels near normal. Testosterone treatment of castrated male or female animals led to a marked increase in enzyme activity with a concomitant steepening of the acinar gradient. Administration of estradiol to normal male rats also led to a reduction in activity, together with a change in the acinar activity gradient. Testosterone treatment of normal females resulted in an induction of PEPCK activity which was most prominent in the periportal zone. The most drastic changes were observed in the perivenous zones. In all experiments a periportal-to-perivenous activity gradient persisted thus marking the periportal zone as the area with highest gluconeogenic capacity.     

The effect of yeast dehydration on the activity of a number of enzymes of cell energetic metabolism

Summary It has been shown that dehydration markedly affects the activity of a number of enzymes connected with energy metabolism in the yeastSaccharomyces cerevisiae. Independently of the drying method used, there was found to be an inverse relationship between the activity of mitochondrial enzymes — NADH-dehydrogenase (EC 1.6.2.1), succinate dehydrogenase (EC 1.3.99.1) and cytochrome C oxidase (EC 1.9.3.1) - and the viability of yeast cells at the stationary growth phase. Dehydration led to an increase in activity only in exogenous NADH-dehydrogenase compared with activity in the initial compressed yeast. On the basis of alcohol dehydrogenase (EC 1.1.1.1) and catalase (EC 1.11.1.6) as examples, an ambivalent effect of the dehydration process on the activity of cytoplasmic enzymes has been demonstrated. The results obtained lead to the conclusion that the activity of individual electron-transport enzymes in yeastSaccharomyces cerevisiae is a sufficiently sensitive to be used as an indicator of the physiological state and to monitor a microbial biomass dehydration procedure.     

Developmental regulation of covalent modification of double-stranded RNA during silkmoth oogenesis

Follicular cells of the silkmoth Bombyx mori contain an enzymatic activity that modifies RNA duplexes in vitro. The modifying activity converts adenosine residues into inosine in duplex but not single-stranded RNA and mediates the partial unwinding of the complement strands. Because of the modification, the RNA loses its ability to form perfect duplexes with its complement upon reannealing in vitro. The modifying enzyme is localized in the cytoplasm of follicular cells and its activity is modulated in a developmentally regulated manner. In contrast, follicular nuclei contain an activity that inhibits the modification and unwinding of duplex RNA. The modifying activity is also present in the cytoplasm of unfertilized oocytes and its accumulation during oogenesis parallels that of the follicular cells. Examination of an established silkmoth cell line of ovarian origin revealed that, in contrast to the situation with follicular cells, the modifying activity has an exclusive nuclear localization. The cytoplasmic fraction of these cells is not only devoid of modifying activity but, as is the case with the nuclear fraction of follicular cells, contains an activity that inhibits duplex RNA modification and unwinding. We conclude that the modification promoting and inhibiting activities are not restricted to a single cell type and that their compartmentalization is developmentally regulated.     

Increased activity of spermidine N1-acetyltransferase in the adrenal cortex of rats following administration of exogenous spermidine, carbamylcholine, 2-deoxyglucose and dopamine agonists

The activity of N1-acetyltransferase was increased in the dissected adrenal cortex of the rat following a single administration of spermidine. The activity was maximal 6-8 h after the onset of treatment. The increase in enzyme activity was abolished when the rats were given cycloheximide 2 h after spermidine; this suggests that increased activity results from an augmentation in the synthesis of the enzyme. Adrenocortical spermidine N1-acetyltransferase was also induced by carbamylcholine, 2-deoxyglucose, apomorphine and piribedil, drugs that are known to cause induction of ornithine decarboxylase in that organ. Hypophysectomized rats showed reduced activity of spermidine N1-acetyltransferase when compared to sham-operated controls, and carbamylcholine no longer elicited an increase in enzyme activity in such animals. Adrenocortical spermidine N1-acetyltransferase activity of hypophysectomized rats is induced by corticotropin (ACTH). These results suggest a hormonal control over the activity of the enzyme in the adrenal cortex with ACTH acting as a mediator.     

Mass correlograms of multiple neuronal activity in the cat's extrastriate cortex

Electrical activity of a population of visually responsive cells located in the vicinity of a single functionally defined neuron was recorded in the area 18 of the cat's cerebral cortex with a single tungsten microelectrode. The correlograms calculated from the mass activity record showed an existence of a rhythmic neuronal firing with an average interval near to 3 ms. When the system was activated by a visual stimulus, a line at an optimal angle moving in an optimal direction, the rhythmic activity became regular, acquiring an oscillatory sinusoidal character. This rhythmic pattern cannot be easily recognized when the activity of a single neuron is recorded. It is possible that such rhythmic activity involving large numbers of neurons contributes to the recognition of the velocity and position of the visual stimulus.     

The ATPase activity of phosphorylase kinase is regulated in parallel with its protein kinase activity.

Phosphorylase kinase from rabbit skeletal muscle has been found to have an intrinsic ATPase activity that occurs at a rate approximately 0.2% of that of its phosphorylase conversion activity and about three times that of its autophosphorylation activity. The characteristics of this ATPase activity were in all aspects tested essentially the same as the kinase's phosphorylase conversion activity. The ATPase requires Mg2+ and is dramatically stimulated by Ca2+ ions. At neutral pH there is a pronounced lag in the rate of product formation that is not present at alkaline pH, a condition that greatly stimulates both the phosphorylase conversion and ATPase activities. ATP is preferentially hydrolyzed over GTP and the Km for MgATP determined in the ATPase assay is 0.14 mM. ADP, an allosteric activator of phosphorylase conversion, also stimulates the ATPase activity, whereas beta-glycerophosphate, an inhibitor of phosphorylase conversion, is an inhibitor of the ATPase activity. Phosphorylation or partial proteolysis of the kinase, which are known to activate phosphorylase conversion, also activate the ATPase activity. Because the phosphorylase conversion and ATPase activities are regulated in parallel, we conclude that activation of the two catalytic activities must share a common underlying basis, namely an enhanced phosphotransferase activity that is independent of the phosphoryl acceptor.     

Involvement of diminution of glutathione, produced by deficiency of methionine in the diet, in the elevation of malic enzyme level in rat liver

Rats fed on a low protein diet show an increase in the specific activity of malic enzyme and a concomitant decrease of glutathione concentration. We have studied the effect on malic enzyme activity of supplementing of low protein diet with essential amino acids. Only when methionine was excluded from the diet did the specific activity of malic enzyme increase to the same extent as found in rats fed with low protein diet. Immunoprecipitation of malic enzyme indicated that specific activity changes are the result of changes in the amounts of enzyme. Under all dietary conditions studied, the increase in malic enzyme activity is associated with a decrease in the concentration of GSH. To evaluate the possible causative role of GSH in malic enzyme induction, the specific activity of malic enzyme was measured in rats treated with BSO, an inhibitor of GSH biosynthesis. The results show that in BSO-treated rats the decrease of GSH levels is also accompanied by an increase in the activity of malic enzyme.     

Light modulation of the activity of protochlorophyllide reductase.

Illumination of etiolated plants effects the activity of protochlorophyllide reductase (NADPH-protochlorophyllide oxidoreductase) in the plastids. Constant illumination or a 2-min light-triggering of etiolated plants leads to an approx. 80% decrease in activity of the enzyme, a change that can be reversed by returning the plants to darkness. The change in activity results from an alteration of the Vmax. rather than Km. Despite the fact that exogenous pigments effect the activity of the enzyme in vitro, no correlation could be drawn between the concentrations of pigments in vivo and activity of the enzyme.     

Histochemical studies of rat lungs after the short-term influence of soil dust

There was examined the biological activity of soil dusts using histochemical methods. Intratracheal administration of dusts used in this study is a common method for testing activity of industrial dusts. The used soil dusts were characterized by high content of free silicon dioxide, 3 times higher than its content in dusts from power stations released in the process of burning coal and approximately to the amount in graphite dust. Both coal dusts and graphite dusts absorbed by the lungs cause silicoanthracosis. The investigations have been shown that soil dusts caused stimulation of the mitochondrial metabolism giving an increase of the activity of succinic dehydrogenase, lactic dehydrogenase, and ATPase induced by Mg++ ions. This was additionally confirmed by an increase of NADP activity which is an enzyme binding a chain or reactions regulating the hydrocarbonic metabolism. There was also observed an increased activity of the hydrolytic enzyme acid phosphatase. High activity occurred in the epithelium of bronchi and bronchioli and focally in pulmonary parenchyma.     

Insulin regulation of lipoprotein lipase in cultured 3T3-L1 cells

Lipoprotein lipase activity is produced by the 3T3-L1 cell an established mouse fibroblast line which resembles an adipocyte after reaching a confluent stage of growth. Since insulin has been shown to be an important regulator of lipoprotein lipase in other mammalian systems, a two hour incubation period was utilized to determine if insulin could enhance an acute response of enzyme activity. Over the range of concentrations tested (0.4, 4.0 and 40 ng/ml), insulin increased lipoprotein lipase activity in acetone ether powders of cells (intracellular enzyme) and the activity secreted into the culture medium. A simultaneous decrease in lipoprotein lipase activity releasable with heparin in a subsequent incubation (membrane bound activity) indicates two distinct effects of insulin on the enzyme in this system.     

Properties of epinephrine-induced activation of cardiac adenosine 3',5'-monophosphate-dependent protein kinase.

The effects of epinephrine on cyclic AMP content and protein kinase activity were examined in an in situ rat heart preparation. Bolus injection of epinephrine into the superior vena cava caused an increase in the activity ratio (-cyclic AMP/"cyclic AMP) of 12 000 X g supernatant protein kinase. The increase was significant within 5 s and maximal in 10 s. Epinephrine produced a dose-dependent increase in both protein kinase activity ratio and cyclic AMP content. The increases in both parameters exhibited a high degree of correlation. The increase in protein kinase activity ratio observed with low doses of epinephrine (less than or equal to 1 microgram/kg) resulted from an increase in independent protein kinase activity (-cyclic AMP) without a change in total protein kinase activity (+cyclic AMP). However, the increase in the activity ratio observed with higher doses of epinephrine (greater than 1 microgram/kg) was due mainly to a decrease in total protein kinase activity rather than a further increase in independent protein kinase activity. The loss of supernatant total protein kinase activity could be accounted for by an increase in activity associated with particulate fractions obtained from the homogenates. A similar redistribution of protein kinase could be demonstrated by the addition of cyclic AMP to homogenates prepared from hearts not stimulated with epinephrine. These results demonstrate that epinephrine over a wide dose range produces a parallel increase in the content of cyclic AMP and the activation of soluble protein kinase. The findings also suggest that protein kinase translocation to particulate material may depend on the degree of epinephrine-induced enzyme activation.     

Marker for real-time analysis of caspase activity in intact cells

Apoptosis, or programmed cell death, is an important regulator of growth, development, defense, and homeostasis in multicellular organisms. A family of cysteine proteases known as caspases is central to many apoptotic pathways, and thus detection of their activity offers an effective means to assess apoptosis. However, currently available methods only allow the evaluation of in vivo caspase activity at a given time point or over a few hours. To assess the activity over extended periods of time, we designed a novel, real-time, in vivo marker that utilizes the N-end rule degradation pathway to allow the detection of caspase activity as reflected by increasing enhanced GFP (EGFP) stability. The marker has an N-terminal arginine in the absence of caspase activity and is rapidly degraded. In vivo caspase activity removes the marker's N-terminal arginine residue, leaving an EGFP with an N-terminal methionine that results in stable fluorescence. In our study, the marker accurately depicted an increase in caspase activity in apoptotic cells and also detected significant endogenous caspase activity in non-apoptotic cells. The downstream effects of this endogenous activity detected in intact, nonapoptotic cells must be regulated by the cell preventing apoptosis. These studies also demonstrate the feasibility of using the N-end rule to study endogenous enzymatic activities other than those associated with proteasomal degradation.     

Effect of water activities of heating and recovery media on apparent heat resistance of Bacillus cereus spores

Spores of Bacillus cereus were heated and recovered in order to investigate the effect of water activity of media on the estimated heat resistance (i.e., the D value) of spores. The water activity (ranging from 0.9 to 1) of the heating medium was first successively controlled with three solutes (glycerol, glucose, and sucrose), while the water activity of the recovery medium was kept near 1. Reciprocally, the water activity of the heating medium was then kept at 1, while the water activity of the recovery medium was controlled from 0.9 to 1 with the same depressors. Lastly, in a third set of experiments, the heating medium and the recovery medium were adjusted to the same activity. As expected, added depressors caused an increase of the heat resistance of spores with a greater efficiency of sucrose with respect to glycerol and glucose. In contrast, when solutes were added to the recovery medium, under an optimal water activity close to 0.98, a decrease of water activity caused a decrease in the estimated D values. This effect was more pronounced when sucrose was used as a depressor instead of glycerol or glucose. When the heating and the recovery media were adjusted to the same water activity, a balancing effect was observed between the protective influence of the solutes during heat treatment and their negative effect during the recovery of injured cells, so that the overall effect of water activity was reduced, with an optimal value near 0.96. The difference between the efficiency of depressors was also less pronounced. It may then be concluded that the overall protective effect of a decrease in water activity is generally overestimated.     

Site-specific mutagenesis of mistletoe lectin: the role of RIP activity in apoptosis.

Recombinant mistletoe lectin (rML) belongs to the class of type II ribosome-inactivating proteins (RIP) composed of a catalytically active A-chain with rRNA N-glycosidase activity and a B-chain with carbohydrate binding properties. To investigate the contribution of the enzymatic activity of the rML A-chain to the observed cytotoxic and apoptotic effects, an rMLA E166Q R169Q molecule was developed by means of site-specific mutagenesis. Following heterologous expression, the activity of mutant rMLA was measured in a cell-free assay for rRNA-N-glycosidase activity. Moreover, after generation of heterodimer, the activities of mutant rML E166Q R169Q and rML wild type were determined in a cytotoxicity and apoptosis assay. Although the reduction of activity as measured in the cell-free RIP assay was more pronounced (factor 237) than in both cellular assays (factors 20-22), the data clearly indicate a close correlation between cytotoxicity, apoptosis, and the enzymatic activity of the rML A-chain. Thus, RIP activity is an essential feature of rML and therefore a prerequisite for its biological function as an anticancer agent.     

Role of an aprotinin-sensitive protease in protein kinase Calpha-mediated activation of cytosolic phospholipase A2 by calcium ionophore (A23187) in pulmonary endothelium

Treatment of bovine pulmonary artery endothelial cells with the calcium ionophore, A23187, stimulates the cell membrane associated protease activity, phospholipase A2 (PLA2) activity, and arachidonic acid (AA) release from the cells. Pretreatment of the cells with arachidonyl-trifluomethylketone (AACOCF3), a cPLA2 inhibitor, but not bromoenollactone (BEL), a iPLA2 inhibitor, prevents A23187 stimulated PLA2 activity and AA release without producing an appreciable alteration of the protease activity. Pretreatment of the cells with aprotinin, an ambient protease inhibitor, prevents the increase in the protease activity and cPLA2 activity in the membrane and AA release from the cells caused by both low and high doses of A23187, and also inhibits protein kinase C (PKC) activity caused by high doses of A23187. Immunoblot study of the endothelial cell membrane isolated from A23187 (10 microM)-treated cells with polyclonal PKCalpha antibody elicited an increase in the 80 kDa immunoreactive protein band along with an additional 47 kDa immunoreactive fragment. Pretreatment of the cells with aprotinin abolished the 47 kDa immunoreactive fragment in the immunoblot. Immunoblot study of the endothelial membrane with polyclonal cPLA2 antibody revealed that treatment of the cells with A23187 dose-dependently increases cPLA2 immunoreactive protein profile in the membrane. It therefore appears from the present study that treatment of the cells with a low dose of A23187 (1 microM) causes a small increase in an aprotinin-sensitive protease activity and that stimulates cPLA2 activity in the cell membrane without an involvement of PKC. By contrast, treatment of the cells with a high dose of 10 microM of A23187 causes optimum increase in the protease activity and that plays an important role in activating PKCalpha, which subsequently stimulates cPLA2 activity in the cell membrane. Although pretreatment of the cells with pertussis toxin caused ADP ribosylation of a 41 kDa protein in the cell membrane, it did not inhibit the cPLA2 activity and AA release caused by both low and high doses of A23187.     

Impact of an Herbicide Combination of Bromoxynil and Prosulfuron on Soil Microorganisms

Soil microcosm experiments were used to investigate the effects on growth and activity of soil microorganisms of an herbicide combination (60% bromoxynil + 3% prosulfuron) frequently used to provide a broad spectrum control of weed species. Culturable aerobic bacteria, fungi, and actinomycetes, the fundamental groups of heterotrophic microorganisms, and nitrifiers, considered a very sensitive group to these compounds, were evaluated. Since herbicides have been found to inhibit decomposition of cellulose in soil, the effects on cellulolytic bacteria and fungi were determined. Dehydrogenase activity as a measure of microbial activity was another parameter considered. The results emphasized a tendency of reversible stimulatory/inhibitory effects of the tested compounds on soil microorganisms, with fungi as an exception. A long-lasting negative action on the activity of the dehydrogenase (DHA), commonly used as an index of the overall microbial activity in soil, was found. The magnitude of these effects were dependent on the assayed concentrations of the herbicides mixture. We concluded that the presence of bromoxynil + prosulfuron could induce significant changes in the microbial populations of the soil, concerning the activity and balance of microbial community. Possible environmental risks must be considered. Dehydrogenase activity was shown to be an important indicator of side-effects attributed to these herbicides.     

Control of phosphatidylcholine synthesis in Hep G2 cells. Effect of fatty acids on the activity and immunoreactive content of choline phosphate cytidylyltransferase.

We examined the effect of fatty acids on phosphatidylcholine synthesis and cytidylyltransferase activity in Hep G2 cells. Treatment of Hep G2 cells with oleic acid caused an increase in the incorporation of [methyl-14C]choline into phosphatidylcholine and a corresponding decrease in radioactivity in choline phosphate using a pulse-chase procedure. This result is consistent with a fatty acid-induced increase in the cytidylyl-transferase step in the choline pathway. We measured cytidylyltransferase activity in membrane fractions and in cytosol (100,000 x g supernatant or soluble enzyme released by digitonin). The activity increased in both membrane and cytosol. Thus, an increase in total activity occurred. Cytidylyltransferase protein determined by Western blot immunoassay increased after oleic acid treatment. Immunotitration of cytidylyltransferase protein also indicated that an increase in enzyme protein resulted from oleic acid treatment. Cycloheximide did not prevent the oleic acid-induced increase in cytidylyltransferase activity. The increase in enzyme activity was apparent when we measured the activity in the presence or absence of lipid activators. Separation of cytosolic cytidylyltransferase into H- and L-forms showed that the increase in cytosolic activity was due to an increase in H-form. The amount of L-form did not change. We interpret these results to suggest that fatty acid treatment of Hep G2 cells promoted the formation of active cytidylyltransferase (H-form) from a preexisting inactive form. The increased activity was distributed between membranes and the lipoprotein form in cytosol (H-form).     

In situ effector pathways of allograft destruction. 1. Generation of the "cellular" effector response in the graft and the graft recipient

Inflammatory leukocytes of DA-to-WF rat renal allografts displayed significant cytolytic activity to natural killer (NK) target cells on Day 2 after transplantation. The NK activity, which was associated with large granular lymphocytes in discontinuous Percoll gradients, peaked on Day 4 and disappeared rapidly thereafter. Coincident with the presence of NK activity in the graft, a decrease in NK activity in the recipient spleen was observed. Low NK activity was also recorded in WF-to-WF autografts. The cells displaying direct cytotoxic activity to donor (but not to recipient) strain peritoneal exudate target cells (PEC) were associated with the T suppressor/killer lymphocytes in affinity chromatography. They appeared in the graft between Days 2 and 4, peaked between Days 6 and 8 and disappeared slowly thereafter. In the spleen the cytotoxic T lymphocyte (CTL) activity appeared later and it reached a maximum between Days 16 and 20 before decreasing. In the blood distinct CTL activity was seen only from Days 16-20 onwards, after the graft had been rejected. No CTL activity was recorded in the graft, blood, or spleen of an autograft recipient. Addition of donor-directed post-transplantation antibody (antibody-dependent cellular cytotoxicity, ADCC) had a slight enhancing effect on the cytotoxic activity of inflammatory leukocytes up to Day 5. After this time, added antibody had a blocking effect on direct CTL activity. No ADCC activity was recorded in the inflammatory population of an autograft. On the contrary, high levels of ADCC activity to donor strain PEC were recorded in the spleens of both autograft and allograft recipients throughout the period of follow-up. The results demonstrate that at least three cellular effector pathways exist in an allograft: a strong natural killer cell component, a strong cytotoxic T lymphocyte component, and (possibly) a weak cell component participating in an ADCC type of cytotoxicity.