Hartmannella culbertsoni: enzymatic, ultrastructural, and cytochemical characteristics of peroxisomes in a density gradient

Isopycnic density gradient centrifugation techniques demonstrated that catalase (EC 1.11.1.6) and urate oxidase (EC 1.7.3.3) had similar distribution patterns with a peak at equilibrium density 1.22 suggesting that both enzymes were associated with a single population of subcellular particles. Catalase (EC 1.11.1.6) was shown cytochemically to be associated with peroxisomes in the sediment of the catalase-rich fractions. Protein showed a bimodal distribution with a soluble peak at density 1.10 and a particulate peak at density 1.20. The particulate protein peak corresponded to the mitochondrial peak. Acid phosphatase (EC 3.1.3.2) had an equilibrium density of 1.10. Acid phosphatase (EC 3.1.3.2) localization and ultrastructural examination of the acid phosphatase-rich fraction revealed that activity was associated with vacuoles. No primary lysosomes were identified.     

Transaminases in rat retina during development

Aspartate, alanine and tyrosine aminotransferase activities have been determined in rat retina during postnatal development. Aspartate transaminase activity is rather low at birth; however, it increases as a function of age, reaching its maximum value at the 23rd day. Alanine transaminase activity increases more rapidly than aspartate transaminase, reaching its maximal value 15 days after birth, but, unlike the latter, this activity considerably decreases during further development. Neonatal tyrosine aminotransferase activity increases by 3 fold between the 5th and 15th day of life.The behaviour of the three enzymes investigated has been related to functional and morphological differentiation of retinal cellular layers.     

Caenorhabditis elegans: Decay of isocitrate lyase during larval development

Changes in the levels of isocitrate lyase, malate synthase, catalase, fumarase, and NADP⁺-isocitrate dehydrogenase have been investigated during larval development of the free-living soil nematode Caenorhabditis elegans in the presence and absence of Escherichia coli. The specific activities of isocitrate lyase, malate synthase, and catalase are maximal at the time of egg hatching and, thereafter, decline during larval development when larvae feed on E. coli, whereas in the absence of E. coli specific activities of the same enzymes increase for 12 hr and subsequently remain constant. There is, however, no change in specific activity of fumarase or NADP⁺-isocitrate dehydrogenase during the same developmental period, in either case. Cycloheximide at 100 μM arrests the decline of isocitrate lyase during development of feeding larvae but has no effect upon the appearance of isocitrate lyase during starvation. The latter is true also for 15 mM itaconate. There is inactivation of isocitrate lyase in crude extracts of frozen worms in comparison to that in analogous extracts prepared from freshly harvested nematodes.     

Trypanosoma brucei: activities and subcellular distribution of glycolytic enzymes from differently disrupted cells

The effect of disruption procedure on the subcellular distribution and the activities of 11 enzymes catalyzing the glycolytic pathway in Trypanosoma brucei has been studied. The activities of the enzymes varied with the lytic procedure used. Maximum specific enzyme activity values were obtained after treatment with saponin whereas digitonin treatment gave the lowest results. The intracellular location of the enzymes was examined by means of differential centrifugation following cell lysis with saponin, Triton X-100, digitonin, or by freezing and thawing. Irrespective of the method of cell lysis employed, the six enzymes, hexokinase, phosphofructokinase, aldolase, phosphoglycerate kinase, glycerol phosphate dehydrogenase, and glycerokinase, were particulate. Of the remaining 5 enzymes, digitonin liberates only phosphoglycerate mutase (partially); saponin or Triton X-100 liberates phosphoglucose isomerase, phosphoglycerate mutase, enolase, and pyruvate kinase but not glyceraldehyde 3-phosphate dehydrogenase; freezing and thawing acts like saponin or Triton X-100 except that it fails to liberate phosphoglucose isomerase, while cell grinding with silicon carbide liberates only glyceraldehyde phosphate dehydrogenase (partially), phosphoglycerate mutase, enolase, and pyruvate kinase. The relative maximal activities of the enzymes suggest that the rate-limiting steps in glycolysis in T. brucei are the reactions catalyzed by aldolase and phosphoglycerate mutase.     

Lipid synthesis and metabolism in the plastid envelope

Plastid envelope membranes play a major role in the biosynthesis of glycerolipids. In addition, plastids are characterized by the occurrence of plastid-specific membrane glycolipids (galactolipids, a sulfolipid). Plant lipid metabolism therefore has unique features, when compared to that of other eukaryotic organisms, such as animals and yeast. However, the glycerolipid biosynthetic pathway in chloroplasts is almost identical to that found in cyanobacteria, and reflects the prokaryotic origin of the chloroplast. Fatty acids generated in the plastid stroma are substrates for a whole set of enzymes involved in the synthesis of polar lipids of plastid membranes such as galactolipids, the sulfolipid, the phosphatidylglycerol. In addition, fatty acids are exported outside the plastid where they are used for extraplastidial polar lipid synthesis (phosphatidylcholine, phosphatidylethanolamine, etc.). Various desaturation steps leading to the formation of polyunsaturated fatty acids occur in various cell compartments, especially in chloroplasts, using fatty acids esterified to polar lipids as substrates. Furthermore, plant glycerolipids can be metabolized by a series of very active envelope enzymes, such as the galactolipid:galactolipid galactosyltransferase and the acyl-galactolipid forming enzyme. The physiological significance of these enzymes is however largely unknown. One of the most active pathways involved in lipid metabolism and present in envelope membranes is the oxylipin pathway: polyunsaturated fatty acids that are released from polar lipids under various conditions (injury, pathogen attack) are converted to oxylipin. Thus, the plastid envelope membranes are also involved in the formation of signalling molecules.     

Studies on metabolism of lung flukes of the genus Paragonimus. VI. Succinoxidase system in homogenates of eggs, larvae, and adults

The present study was concerned with the succinoxidase system in Paragonimus westermani, Paragonimus ohirai, and Paragonimus miyazakii. Potassium cyanide inhibited the motility of larval and adult forms. Succinate stimulated the reduction of methylene blue by homogenates of embryonated eggs, larvae, and adults, while malonate inhibited the reduction. Reduced cytochrome c was oxidized by the 1,000g supernatant from homogenates of embryonated eggs, larvae, and adults. The supernatant prepared from unembryonated eggs did not oxidize reduced cytochrome c. Succinate stimulated oxygen consumption by the homogenate of adult worms. Oxygen consumption markedly increased in the homogenate of adults when both succinate and cytochrome c were added as substrate to the reaction mixture, while malonate and cyanide inhibited oxygen consumption.     

Plagiorchis elegans: Histochemical localization of dehydrogenases in the cercarial stage

Cercariae of Plagiorchis elegans Rudolphi 1802 collected from experimentally infected snails, Lymnaea palustris, were subjected to various histochemical tests for dehydrogenase systems. A high degree of activity was demonstrated for succinic dehydrogenase (EC 1.3.99.1), malic dehydrogenase (EC 1.1.1.37), isocitric dehydrogenase (EC 1.1.1.41), α-glycerophosphate dehydrogenase (EC 1.1.1.8), and glucose 6-phosphate dehydrogenase (EC 1.1.1.49). These enzymes were present in the tegument, tail, caudal pocket, excretory bladder, acetabulum, and oral sucker, particularly in the muscles around the stylet. Only moderate activity was obtained for lactic dehydrogenase (EC 1.1.1.27) and 6-phosphogluconate dehydrogenase (EC 1.1.1.44) at these sites, glutamic dehydrogenase (EC 1.4.1.2) was localized only in the tails of the cercariae and tests for alcohol dehydrogenase (EC 1.1.1.1) were completely negative. The cerebral ganglia and its commissures stained intensely in the tests for succinic, isocitric, α-glycerophosphate, and glucose 6-phosphate dehydrogenase systems. The results indicate the possibility that several energy-producing sequences may be available to these cercariae.     

Ascaris lumbricoides: Coproporphyrinogen oxidase activity in eggs and muscle

The possible presence of coproporphyrinogen oxidase (EC 1.3.3.3), an oxygen-requiring enzyme in the porphyrin biosynthetic pathway, was investigated in supernatant fractions of homogenized Ascaris lumbricoides muscle and developing eggs, and in mitochondrial preparations of muscle. Compared with rat liver controls, low levels of enzyme activity were found in A. lumbricoides gut, 6-day eggs, and muscle mitochondria. Enzyme activity in muscle, 8-day, and 25-day eggs was not measurable under the conditions employed.     

T4 and ultrasensitive TSH immunoassays using luminescent enhanced xanthine oxidase assay

The use of xanthine oxidase in immunoanalysis has never been reported. We describe here a procedure in which the xanthine oxidase dependent luminescence of luminol is enhanced in the presence of Fe–EDTA complex, providing an highly sensitive assay (3 amol of enzyme) and a long-term signal. This specific amplification has been applied to T₄ and ultrasensitive TSH solid phase immunoassays, with T₄–XO and anti-TSH monoclonal antibody-XO conjugates as tracers. The performances of these assays are at least equivalent to those obtained with iodinated tracers, using the same solid phases and the same calibrators. The major advantages of these immunoassays are: (1) the long-term signal which can be repeatedly recorded over several days, (2) the high detection sensitivity, (3) the long-term stability of the luminescence reagent and (4) the stability of the conjugates.     

Caenorhabditis elegans and Ascaris suum: fragmentation of isocitrate lyase in crude extracts

It is well known that proteolysis often occurs after rupture of metazoan cells. Thus proteins isolated from extracts may not be representative of their native cellular counterparts. In the present research, extensive proteolysis was observed in crude extracts of the freeliving soil nematode Caenorhabditis elegans and the parasitic nematode Ascaris suum. Phenylmethylsulfonyl fluoride (PMSF) reduced the loss in activity of isocitrate lyase (EC 4.1.3.1), fumarase (EC 4.2.1.2), and citrate synthase (EC 4.1.3.7) in extracts of C. elegans but had little or no effect upon loss of malate synthase (EC 4.1.3.2). Catalase (EC 1.11.1.6) was stable. The loss of isocitrate lyase and citrate synthase was less pronounced in extracts of 22-day-old embryos of A. suum. Catalase decayed in these extracts. The addition of PMSF reduced the loss in all three of these activities. Fumarase was stable. The number of active fragments of isocitrate lyase recovered after filtration on Sephadex G-200 increased with the length of storage of crude extracts in the absence of PMSF at 4 C. Even in the presence of PMSF five activity peaks were observed after storage of extracts of C. elegans at 4 C for 72 hr. The molecular weights of active species ranged between 549,000 and 128,000 for isocitrate lyase in extracts of either C. elegans or A. suum. The 549,000- and 214,000-dalton species of isocitrate lyase from A. suum were much more labile at 50 C than the 543,000- and 195,000-dalton species from C. elegans.     

Schistosomatium douthitti: carbohydrate metabolism in the adults.

Pathways of carbohydrate metabolism in the adults of Schistosomatium douthitti: were investigated. Histochemical reactions for adenosinetriphosphatase (EC 3.6.1.3) glucose 6-phosphate dehydrogenase (EC 1.1.1.49), phosphogluconate dehydrogenase (EC 1.1.1.43), glycerol-3-phosphate dehydrogenase (EC 1.1.1.8), lactate dehydrogenase (EC 1.1.1.27, 1.1.2.3) isocitrate dehydrogenase (EC 1.1.1.41), succinate dehydrogenase (EC 1.3.99.1), malate dehydrogenase (EC 1.1.1.37), cytochrome oxidase (EC 1.9.3.1), and adenosine triphosphatase (EC 3.6.1.3) were found in the adult worms. Glycogen deposits occurred in the parenchyma.Low oxygen tension immobilized the worms. Tartar emetic, sodium cyanide reduced adult motility in vitro. Manometric experiments demonstrated a respiratory quotient of approximately one. Oxygen uptake was completely inhibited by tartar emetic and partially inhibited by sodium fluoracetate and sodium cyanide. Inhibition by sodium fluoroacetate was partially counteracted by citric acid in the medium.Adults demonstrated an oxygen debt following anaerobic incubation. A maximum of 52% of the glucose consumed under aerobic conditions was excreted as lactic acid. Under anaerobic conditions the amount of lactic acid excreted increased. Acids other than lactic acid were also released. Results indicate that although glycolysis is the major pathway, two additional aerobic pathways also exist, one which is cyanide sensitive and the other cyanide insensitive.     

Entamoeba histolytica: ultrastructural localization of Ca2+-dependent nucleotidases

Localization of nucleotidases dependent on Ca²⁺ was investigated cytochemically in axenically cultivated trophozoites of Entamoeba histolytica, strain HM-1:IMSS, with an electron microscope. Ca²⁺-dependent ATPase (EC 3.6.1.3) activity was found on the plasma membrane and on the inner surface of the limiting membrane of a few cytoplasmic vacuoles. Ca²⁺-dependent ADPase, Ca²⁺-dependent thiamine pyrophosphatase, and acid phosphatase (EC 3.1.3.2) activities were detected on the inner surface of the limiting membrane of most of the cytoplasmic vacuoles but not on the plasma membrane. Cytoplasmic vacuoles with these enzymatic activities seemed similar in morphological characteristics. Moreover, the reaction product formed by Ca²⁺-dependent ADPase, Ca²⁺-dependent thiamine pyrophosphatase and acid phosphatase was demonstrable on the inner surface of the limiting membrane of vacuoles containing ingested red blood cells. The reaction product formed by these enzymes was also observed on the periphery of ingested red blood cells. The findings suggest that cytoplasmic vacuoles with these enzymatic activities are lysosomal in nature, probably phagolysosomes; therefore, the enzymes appear to be at least partially associated with primary lysosomes of E. histolytica.     

Receptor modifiers indicate that 4-aminobutyric acid (GABA) is a potential modulator of ion transport in plants

GABA (4-aminobutyric acid) is a ubiquitousnon-protein amino acid that accumulates rapidly inplants in response to stress. GABA was firstidentified in plants (potato tubers) and animals(brain tissue) 50 years ago. Although GABA is nowrecognized as the most important inhibitoryneurotransmitter in the mammalian central nervoussystem (CNS), the role of GABA in plants remainsunclear. Studies were performed using Lemna toinvestigate the possibility that GABA elicits aresponse in plants that may be related to that of asignaling molecule as described for GABA effects onthe CNS. Lemna growth was increased 2 to 3-foldby 5 mM GABA, but growth was strongly inhibited by 0.5mM of the isomers 3-aminobutyric acid and2-aminobutyric acid. Growth promotion by GABA wasrapidly terminated by addition of 2-aminobutyric acidto the culture medium, but inhibitory effects of2-aminobutyric acid were not reversed by GABAregardless of amounts added. Promotion of Lemnagrowth by GABA was associated with an increase inmineral content of treated plants in a dose dependentmanner. Results support the hypothesis that GABAactivity in plants involves an effect on ion transportand an interaction with a receptor. Evidence for GABAreceptors in Lemna was obtained from experimentswith pharmacological agents that have been used toidentify GABA receptors in animals. GABA mediatedpromotion of Lemna growth was inhibited bybicuculline and picrotoxin, which are respectivelycompetitive and non-competitive antagonists of GABAreceptors in the CNS. Growth inhibition bybicuculline was not relieved by increasing the amountsof GABA in the medium, indicating that the alkaloid isnot acting, as in the CNS, by competitive antagonismof GABA at GABA receptor sites. Baclofen, a GABAagonist that promotes GABA activity in animalssignificantly increased GABA mediated promotion ofLemna growth. These findings and the knownaction of GABA in regulating ion channels in animalssuggests a way that GABA could amplify the stressresponse in plants.     

Alkaline and acid phosphatase in the intestines of rats infected with a virulent strain of Entamoeba histolytica and treated with emetine and Flagyl

The distribution patterns of alkaline phosphatase (EC 3.1.3.1) and acid phosphatase (EC 3.1.3.2) in the intestine of rats inoculated intracaecally with a virulent strain of Entamoeba histolytica and treated with emetine hydrochloride and metronidazole (Flagyl) were studied. The caecum and the large intestine showed a highly significant increase in alkaline phosphatase activity after amoebic inoculation, and the enhanced activity was lowered by emetine and Flagyl treatment. There was no significant increase in acid phosphatase activity either in the caecum and the large intestine or in the small intestine (ileocaecal end). Intracaecal inoculation of bacterial associates alone from E. histolytica cultures did not produce any significant change in the level of these enzymes in the intestine.     

A histochemical study of the innervation of cerebral blood vessels in the snake

Summary Dual innervation of snake cerebral blood vessels by adrenergic and cholinergic fibres was demonstrated with the use of histochemical methods. Although the nerve plexuses are somewhat less dense, the essential features of innervation of the blood vessels are similar to those of mammals with the exception that the adrenergic plexuses are more prominent than the cholinergic plexuses. The major arteries of the cerebral carotid system have a rich nerve supply. However, the innervation is less rich in the basilar and poor in the spinal (vertebral) arteries. Although the arteries supplying the right side of head are poorly developed, three pairs of arteries, cerebral carotids, ophthalmics and spinals, supply the snake brain. The carotids and ophthalmics are densely innervated and are accompanied by thick nerve bundles, suggesting that the nerves preferentially enter the skull along those arteries. Some parenchymal arterioles are also dually innervated. Connection between the brain parenchyma and intracerebral capillaries via both cholinergic and adrenergic fibres was observed. In addition cholinergic nerve fibres, connecting capillaries and the intramedullary nerve fibre bundles, were noticed. Capillary blood flow may be influenced by both adrenergic and cholinergic central neurons. The walls of capillaries also exhibit heavy acetylcholinesterase activity. This may indicate an important role for the capillary in the regulation of intracerebral blood flow.     

Caenorhabditis elegans and Panagrellus redivivus: Enzyme-mediated modification of chemotaxis

Treatment with mannosidase or sialidase completely inhibited chemotactic responses of Caenorhabditis elegans wild type, C. elegans mutants CB1377 (daf-6)X and CB1379 (che-3)I, and Panagrellus redivivus to a source of attractants. Trypsin (EC3.4.21.4) caused a partial reduction in the level of chemoresponse. Normal chemotaxis was renewed within 20 hr following exposure to the enzymes. Other enzymes tested had no effect. Experimental and supporting evidence is presented that behavioral modification resulted from functional impairments to receptors located within chemosensory sensilla.     

Thermosensitive respiratory deficiency in yeast associated with specific effects on particulate cytochrome oxidase.

A mutant of Saccharomyces cerevisiae, unable to grow at the expense of non fermentable carbon sources at 37 degrees C, has been selected; at 25 degrees C the mutant strain behaves like the parental wild strain. Evaluations of respiration rates during aerobic growth at restrictive temperature on one hand, enzymatic and/or spectral evaluations of the individual components of the respiratory chain on the other hand show that the respiratory deficiency is specifically correlated with a reduced level of cytochrome oxidase. The decrease of enzyme activity is the direct consequence of a lowering of hemoprotein (a,a3) concentration. Temperature-activity relationship of cytochrome oxidase elaborated at the permissive temperature by the mutant strain is modified as far as the particulate enzyme is concerned, but no difference is observed after partial solubilization of the enzyme by non ionic surfactant. Genetic analysis shows that the mutant phenotype results from a nuclear gene mutation.     

Calorimetric investigation of NAD binding to some dehydrogenases.

The thermodynamic parameters for the binding of NAD to some dehydrogenases have been determined calorimetrically at 25° and pH 7.6. Except for liver alcohol dehydrogenase (LADH) the ΔG^o, ΔH^o and ΔS^o values for NAD binding to the dehydrogenases are very similar pointing out a possible structure - thermodynamics correlation. The large deviation observed in the case of LADH would be consistent with the occurrence of a conformational change in this enzyme upon binding NAD.     

Cytoplasmic free calcium concentration in porcine platelets: Regulation by an intracellular nonmitochondrial calcium pump and increase after thrombin stimulation

Mechanisms are assumed to exist in the resting platelet which maintain the concentration of cytoplasmic free calcium below that level required to activate cellular responses. To assess such processes the porcine platelet plasma membrane was selectively lysed with digitonin and the uptake (or flux) of free calcium monitored by an extracellular calcium electrode. Lysis resulted in an immediate lowering of the extracellular free calcium, due to the action of intracellular organelle(s) acting on the extracellular space through the permeabilized plasma membrane. In resting platelets, the rate of calcium uptake was first order with respect to the extracellular prelytic calcium concentration, and hence the cytoplasmic free concentration was found to be 1·10^?7 M by extrapolation to a point of zero flux (i.e., the null point). This approach could not be used with thrombin-stimulated platelets, as external calcium was required for both secretion of ATP + ADP and aggregation. Nevertheless, evidence for an increase in cytoplasmic free calcium after thromin stimulation was obtained. Metabolic inhibitors and agents known to inhibit calcium uptake by mitochondria had no effect on the calcium flux following lysis, indicating different mechanisms for calcium homeostasis in the platelet when compared with other cell types (e.g., liver). Levels of ionophore A23187, which caused platelet aggregation, gave a massive release of the nonmitochondrial pool of calcium into the cytoplasmic space. Thus, in porcine platelets an intracellular energy-requiring calcium pump, which sequesters calcium in a nonmitochondrial membranous compartment, is crucial for intracellular calcium homeostasis.