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.     

Possible correlations between the level of alcoholic motivation and the electrophysiological structure of sleep in the rat

Using modified Porsolt's method, the electrophysiological sleep pattern was studied in normal conditions and after a single intraperitoneal ethanol injection to noninbred male albino rats divided into 2 groups ("high activity" and "low activity" rats). Voluntary alcohol intake in these rats was measured during free choice between 10% ethanol and water for 20 days. "Low activity" rats were characterized by a statistically significant 3.4-fold higher level of ethanol consumption and 2.7-fold longer REM-sleep stage, as compared to "high activity" animals. In "low activity" animals ethanol (1 g/k, 10% solution, i. p.) inhibits and in "high activity" rats it increases REM-sleep stage, thus removing differences in the sleep pattern in the two groups of rats. The data obtained suggest a possible role of REM-sleep in the development of alcohol motivation.     

Mutational analysis of the "regulatory module" of hormone-sensitive lipase

Hormone-sensitive lipase (HSL) is a rate-limiting enzyme in lipolysis that displays broad substrate specificity. HSL function is regulated by reversible phosphorylation that occurs within a 150 aa "regulatory module" of the protein. The current studies used mutational analysis to dissect the contribution of the "regulatory module" in HSL activity and substrate specificity. Deletion of the entire "regulatory module" or replacement of the "regulatory module" with the "lid" of lipoprotein lipase resulted in enzymatically inactive proteins. Deletion of sequentially longer stretches of the "regulatory module" resulted in a stepwise reduction in hydrolytic activity. Analysis of 7-19 amino acid deletional mutants that spanned the "regulatory module" showed that the N-terminal partial deletion mutants retained normal hydrolytic activity and activation by PKA. In contrast, the C-terminal partial deletion mutants displayed reduced hydrolytic activities, with preferential loss of activity against lipid-, as opposed to water-soluble, substrates. Single amino acid mutations of F650C, P651A, and F654D reduced activity against lipid-, but not water-soluble, substrates. The current results suggest that the length of the "regulatory module" and specific sequences within the C-terminal portion of the "regulatory module" of HSL (amino acids 644-683) are crucial for activity and appear to be responsible for determining lipase activity.     

N-acetyltransferase activity during growth of the transplantable Pliss lymphosarcoma and during pregnancy in rats

Study of the distribution of female noninbred rats according to N-acetyltransferase activity has permitted the conclusion to be derived that the animals may have a "slow", "intermediate" and "fast" phenotype of acetylation. It was discovered that the rate of N-acetyltransferase activity increased 1.5-2-fold on days 12 and 15 after transplantation of Pliss's lymphosarcoma. The time course of changes in N-acetyltransferase activity was characterized by individual features. The pattern of changes in N-acetyltransferase activity in pregnancy was dependent on the initial acetylation phenotype: in animals with a "slow" and "intermediate" phenotype of acetylation, the activity ascended by the 21st day of pregnancy, whereas in animals with a "fast" phenotype of acetylation, it declined.     

Intra- and extracellular forms of lipoprotein lipase in adipose tissue.

The location of lipoprotein lipase activity in rat adipose tissue was studied using intact epididymal fat pads, isolated adipocytes, and lipoprotein lipase activity secreted from adipocytes as enzyme sources. The enzyme activities of these preparations were characterized by gel filtration. The method used for isolation of adipocytes had been modified to minimize activation of lipoprotein lipase during the procedures. Extracts of intact adipose tissue separated into two major lipoprotein lipase activity peaks, designated "a" and "b", the "a" fraction representing about 30 (fasted rats) to 50% (fed rats) of the total enzyme activity. An intermediate fraction (designated "i") was frequently observed. Extracts of isolated adipocytes from fed rats contained about 35% and those from fasted rats about 65% of the lipoprotein lipase activity present in intact tissue. The "b" fraction constituted 80--97% of the adipocyte lipoprotein lipase activity. In contrast, the enzyme activity secreted from the adipocytes contained only the "a" and "i" fractions. These data implicate the existance of one intracellular form of lipoprotein lipase (corresponding to the "b" fraction), different from extracellular forms of the enzyme (corresponding to fractions "a" and "i"). A transformation of the intracellular to the extracellular forms appears to occur in conjunction with secretion of enzyme from the fat cell.     

Task Relevance Enhances Early Transient and Late Slow-Wave Activity of Distributed Cortical Sources

The primary purpose of these studies was to link together concepts related to attention/working memory and feedforward/feedback activity using MEG response profiles obtained in humans. Similar to recent studies of attention in monkeys, we show early spike-like activity (<200 ms poststimulus), most likely reflecting an early transient excitatory response mixed with feedback influences, followed by slow-wave activity (>200 ms poststimulus) in MEG cortical response profiles evoked by a visual working memory task. We experimentally dissociated the early transient activity from the later sustained activity (predominately feedback) by conducting an auditory size classification task. Words, representing common objects, evoked activity in occipital cortex (presumably due to imagery) even though visual stimuli were not present in this task. The initial spike was absent from the response profile obtained from occipital cortex, leaving only slow-wave activity, thereby allowing us to characterize or profile feedback activity in this situation. Attention or task relevance enhanced the initial spike and slow-wave activity in visually responsive areas. Prefrontal activity, along the superior frontal sulcus, evoked by the working memory task, was active later in time than initial activity in visual cortex and later than the earliest effect of attention modulation in visual cortex.     

The Activities of Six Exo- and Endopeptidases in the Substantia Nigra,Neostriatum, and Cortex of the Rat Brain

We determined the enzymatic activity and crude subcellular distribution of four exopeptidases: Dipeptidylaminopeptidase IV (DAP-IV), Alanyl aminopeptidase (AAP), Prolyl aminopeptidase (PAP) and -Glutamyl transpeptidase (GTP), and two endopeptidases: Postproline endopeptidase (PEP) and Trypsin-like peptidase (T-L P) in pars compacta (SNPC) and pars reticulata (SNPR) of substantia nigra, caudate-putamen (CAU) and cerebral cortex (CC) of the rat brain. We found: 1) DAP-IV activity is comparatively higher in SNPC and it is equally distributed in the postmitochondrial precipitate (PR) and supernatant (SN) fractions of SNPC, CAU and CC but higher in the SN from SNPR. 2) CC shows the highest activity of AAP and its activity is mainly located in the SN from all areas. 3) The activity of PAP is comparatively higher in SNPC and it is exclusively located in the SN from all areas. 4) GTP activity is similar in all areas but its predominance is in the SN for SNPC and SNPR, and in the PR for CAU and CC. 5) CAU has higher PEP activity (higher in the PR) than CC (higher in the SN); no activity is detected in the substantia nigra. 6) The activity of a Trypsin-like peptidase is the highest in SNPC and SNPR; this activity have some predominance in the SN and higher predominance in the same fraction from CAU and CC.     

Genetic control of developmental patterns of cerebral enzyme activities: Further differences between C3H and ICR strains of mice

We have investigated developmental changes in activity for five enzymes associated with different cerebral metabolic systems in two separate strains of mice. The enzymes studied were acid -galactosidase, arylsulfatase A, cerebroside -galactosidase, cerebroside sulfotransferase, and glutamate decarboxylase. The two strains of mice were C3H/SWV and ICR/SWV. We confirm the experiments of Meisler, Paigen, and colleagues showing higher acid -galactosidase activity throughout development in C3H mice. In addition we have demonstrated higher arylsulfatase A activity throughout development in C3H mice. The shape of the developmental curve for arylsulfatase A activity in brain in the two strains was similar. There were no differences in developmental changes of activity between the two strains for the other three enzymes studied.     

The increase in activity of acid hydrolases in muscles of rats after subcutaneous administration of dimethyl-para-phenylene diamine. A combined histochemical and biochemical investigation

Summary The increase in activity of acid hydrolases in skeletal muscles of rats after subcutaneous administration of dimethyl-para-phenylene diamine (DPPD) was studied with a combined histochemical and biochemical investigation. In part I the histochemical findings were presented. In this communication the biochemical findings are reported and compared with the histochemical findings.In homogenates of m. biceps femoris, m. gastrocnemius and m. rectus femoris of DPPD-treated rats, the activity of the lysosomal acid hydrolases, cathepsin D, acid maltase, acid phosphatase, and -glucuronidase was increased. This increase in activity was maximal after 7 to 9 days of DPPD treatment and ran parallel to the severity of the pathological changes. Statistical calculations clearly reveal that the increased activity of one acid hydrolase was significantly paralleled by an increased activity of a second acid hydrolase. Moreover these calculations reveal that the biochemical activity findings correlated with the histochemical activity findings. However it was remarkable that in the histochemical study, the estimated increase in acid phosphatase activity was much more than the increase in acid phosphatase activity found biochemically, whilst on the other hand the histochemically estimated increase in -glucuronidase activity corresponded with the biochemical observations. The results of gel filtration techniques have shown that this discrepancy of acid phosphatase activity was caused by different substrate specificity of the different isoenzymes of acid phosphatase and that as a result of the DPPD treatment the isoenzyme pattern had been altered. The elution patterns showed three distinct isoenzymes of acid phosphatase of normal and of DPPD treated rats. These isoenzymes, termed I, II and III, have molecular weights of: 200,000 or more, 83,500–104,500 and 14,500–18,100. Isoenzymes I and II split the substrates 4-methylumbelliferyl phosphate and naphthol AS-BI phosphate and the activity is strongly increased in the muscles of the DPPD treated rats. Isoenzyme III does not split naphthol AS-BI phosphate and the activity is not increased in the muscles of the DPPD treated rats. Considering the fact that it has been shown that the activity of isoenzyme III is high compared with that of the isoenzymes I and II, it is important to realise that by using naphthol AS-BI phosphate not all acid phosphatase can be demonstrated in sections of skeletal muscle.This study was partly supported by a grant from the Prinses Beatrix Fonds, 's Gravenhage, The Netherlands, and was mainly extracted from the Ph. D. thesis of D.E. Israël (1977).     

The cytochrome oxidase activity of the nuclei and nuclear membranes of rat liver cells following partial hepatectomy

The cytochrome oxidase activity of liver cell nuclei determined in the presence of ascorbate and cytochrome c equals, on the average, 1.2 nmol O2/min/mg protein. This activity tentatively named "free" cytochrome oxidase activity is in nuclei 10% and in nuclear envelopes about 30% of the total cytochrome oxidase activity determined in the presence of tetramethyl paraphenylendiamine. Following the partial hepatectomy, the "free" cytochromoxidase activity in the isolated nuclei is 5-6 times that in the control and approaches the total cytochromoxidase activity which remains at the same level. No increase of free cytochrome oxidase activity in the rat liver homogenate and the isolated mitochondria following the partial hepatectomy was observed.     

Analysis of postinspiratory activity of phrenic motoneurons with chemical and vagal reflexes

We examined the effects of chemical and reflex drives on the postinspiratory inspiratory activity (PIIA) of phrenic motoneurons using a single-fiber technique. Action potentials from "single" fibers were recorded from the C5 phrenic root together with contralateral mass phrenic activity (also from C5) in anesthetized, paralyzed, and artificially ventilated cats with intact vagus and carotid sinus nerves. Nerve fibers were classified as "early" or "late" based on their onset of discharge in relation to mass phrenic activity during hyperoxic ventilation. Only the early fibers displayed PIIA but not the late fibers, even when their activity began earlier in inspiration with increased chemical drives. Isocapnic hypoxia increased, whereas hyperoxic hypercapnia shortened the duration of PIIA. Pulmonary stretch and "irritant" receptors inhibited PIIA. Hypercapnia and stimulation of peripheral chemoreceptors by lobeline excited both early and late units to the same extent, but hypoxic ventilation had a less marked excitatory effect on late fiber activity. Irritant receptor activation increased the activity of early more than late fibers. Hyperoxic hyperventilation eliminated late phrenic fiber activity, whereas early fibers became tonically active. Bilateral vagotomy abolished this sustained discharge in eight of nine early units, suggesting the importance of vagal afferents in producing tonic firing during hyperventilation. These results suggest that early and late phrenic fibers have different responses to chemical stimuli and to vagally mediated reflexes; late units do not discharge in postinspiratory period, whereas early fibers do; the PIIA is not affected in the same way by various chemical and vagal inputs; and early units that exhibit PIIA display tonic activity with hyperoxic hypocapnia.     

畜禽粪便堆肥过程中酶活性及微生物数量的变化研究

实验选取鸡粪和猪粪进行好氧堆肥发酵,研究畜禽粪便腐熟过程中酶活性和微生物的变化趋势以及相互联系。结果表明:过氧化氢酶活性和纤维素酶活性在堆肥初期较高,随后迅速降低,最终过氧化氢酶维持在9~12ml/g之间,纤维素酶维持在12.37~15.07mg/(kg·h)之间,而脲酶活性变化趋势为"升高-降低-升高"。细菌数量变化趋势为"低-高-低";放线菌为"高-低";真菌为"高-低-高"。通过相关分析发现,放线菌可能是影响堆肥中过氧化氢酶和纤维素酶的关键因素。鸡粪中放线菌与过氧化氢酶呈极显著正相关;猪粪中放线菌与过氧化氢酶和纤维素酶呈显著正相关;鸡粪+猪粪中放线菌与过氧化氢酶和纤维素酶呈极显著正相关。     

Activity of lumbosacral interneurons during fictitious scratching

Activity of lumbosacral spinal interneurons was studied during fictitious scratching in decerebrate, immobilized cats. Neurons whose activity changed during fictitious scratching were located in the substantia intermedia lateralis and ventral horn. Among these neurons cells were distinguished whose activity was modulated in rhythm with motor discharges to different muscles (61.6%) and cells which were activated tonically (21.4%) or inhibited tonically (17%). By correlation of activity with discharges to corresponding muscles the rhythmically activated neurons were divided into "aiming" (36.6%) and "scratching" (25%). Neurons whose activity was unchanged during fictitious scratching also were observed. These cells were located mainly in the more dorsal regions of gray matter. Neurons to which wide convergence of excitatory influences from high-threshold cutaneous and muscular afferents was observed were mainly placed in the "aiming" group. "Scratching" neurons, compared with "aiming," more often received inputs only from low-threshold cutaneous or high-threshold muscular afferents. Group Ia interneurons were activated in phase with the corresponding motoneurons. Passive displacement of the limb in a forward direction predominantly inhibited spike activity of the "aiming" and potentiated activity of the "scratching" neurons. The neuronal organization of the spinal scratch generator is discussed on the basis of the results.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 13, No. 1, pp. 57–66, January–February, 1981.     

A comparative study in young male animals of 10 species of the distribution of alkaline phosphatase activity in small arteries

Summary The distribution of alkaline phosphatase activity in the walls of small arteries measuring 100–200 in internal diameter in the collapsed state was investigated in nine organs in male animals of each of ten species employing the Gomori and azo-coupling techniques. Alkaline phosphatase activity was high in relation to the arterial endothelium of the chick, hen, turkey, rabbit, frog and man but was related to the arterial adventitia and not to the endothelium in the rat. In the guinea pig and hamster arterial alkaline phosphatase activity was uniformly low or absent. Within nine of these ten species the pattern of organ distribution of arterial alkaline phosphatase activity in vessels of comparable size was uniform. In the cat however, enzyme activity was confined to the adventitia of small cerebral arteries and to the media of the smallest arteries (arterioles) in all organs except the testis from which arterial activity was absent.The distribution of alkaline phosphatase activity in 100–200 , arteries in the species investigated was not directly related to the presence of capillary endothelial activity which was detected in all the organs examined. The activity in the walls of the smallest arteries (arterioles) was located in different sites in different species.United Arab Republic Research Fellow.     

Supermolecular organization of aminotransferases and dehydrogenases of alpha-ketoacid from rat brain mitochondria

A fraction of coarse mitochondria from the rat brain was deposited after short-term effect of supersound to obtain "metabolones". Activity of dehydrogenases of alpha-ketoacids, succinate dehydrogenase, aspartate-, alanine- and GABA-alpha-ketoglutarate amino-transferases has been determined in the supernatant liquid and in "metabolones". It is shown that dehydrogenase activity is mainly (93-100%) localized in "metabolones", while the level of aminotransferase activity in the latter is lower (72-94%). Nonproportionally high activity of aminotransferases in the supernatant liquid is found to considerably suprpass a decrease in activity of these enzymes in "metabolones" against a background of extremely scanty losses of protein (within 5%) induced by the supersound effect. A hypothetic model of a "metabolone" containing the enzymes of the Krebbs cycle and GABA-shunt is suggested.     

Inhibition of catalase activity by oxidative stress and its relationship to salicylic acid accumulation in plants

The decrease in catalase activity and its relationship to change in salicylic acid content were investigated in rice, wheat, and cucumber seedlings exposed to oxidative stresses. A decrease in chlorophyll fluorescence (F/Fm), measured as an indicator of the oxidative stress, and a drop in catalase activity were observed following treatment with NaCl in all plant seedlings tested . Furthermore, such decreases in F/Fm and catalase activity were also observed under low temperature conditions in both rice cultivars, whereas the degrees of decrease were dependent on their low temperature tolerance . Although the content of salicylic acid increased in rice seedlings stressed by NaCl treatment, it was inversely correlated with the decrease in the catalase activity . Such a relationship between the decrease in catalase activity and increase in salicylic acid content was confirmed with paraquat treatment of the rice seedlings . These results suggested that the fall in catalase activity is a phenomenon occurring in many plant species under oxidative stress and is related to the accumulation of salicylic acid in oxidatively-stressed plants.     

Effect of altered hormonal states on the histochemical distribution of lipase activity in the rat prostatic complex

Summary Lipase activity is localized mainly in the cytoplasm of the epithelial cells of all the rat prostatic lobes, and in periacinar stroma of the dorsal prostate; lateral lobe has little enzyme activity. Progesterone (1 and 2.5 mg) causes an increase in lipase activity in the acinar epithelia, periacinar and interfollicular stroma, blood vessels and the mast cells of the ventral prostate. A low dose of estrogen (0.1 g) markedly stimulates and a high dose (5 g) virtually obliterates the activity of the enzyme in the prostatic complex. Following cessation of progesterone therapy the enzyme activity is augmented still further, but near normalization is seen after withdrawal of estrogen. When the two steroids are given conjointly, an estrogenic pattern of morphology is the feature; at the same time there is some suggestion of an antagonism between the two hormones with respect to their effect on the enzyme. Thyroidectomy causes an increase in lipase activity of the complex which is not normalized after thyroxine therapy.Communication no. 1398. Part IX in the series Prostate and endocrines.     

Age-related changes of glucose-6-phosphate dehydrogenase activity in mouse oocytes

Summary Glucose-6-phosphate dehydrogenase (G6PDH) activity was measured in follicular oocytes and in ovulated eggs of prepubertal, adult and aged mice. G6PDH activity in ovulated eggs was 60% of the activity in follicular oocytes in all age groups. The mean G6PDH activity was significantly higher in follicular oocytes of adult mice than in oocytes of both prepubertal and aged mice. In aged mice, the decreased mean activity in follicular oocytes as well as in ovulated eggs was mainly due to a high percentage of cells with extremely low activity (25 and 18%, respectively). The percentage of preovulatory oocytes with low activity in prepubertal mice was 9% and in adult mice 0.3%. For ovulated eggs these percentages were 0% for both prepubertal and adult mice. In every age group, all ovulated eggs showed a normal morphology. When ovulated eggs with extremely low G6PDH activity can still be fertilized, it can be questioned whether this loss of activity could cause disturbances in development of (preimplantation) embryos. Our findings emphasize the potentialities of investigating intact single oocytes for changes in enzyme activities, which could be applied as parameters for quality control of these cells.     

Regulating effect of mitochondrial lactate dehydrogenase on oxidation of cytoplasmic NADH via an "external" pathway in skeletal muscle mitochondria.

1. The specific activity of lactate dehydrogenase of skeletal muscle mitochondria was found to be 2.5 times lower than specific activity of total NADH-cytochrome c reductase. 2. The specific activity of mitochondrial LDH in skeletal muscle mitochondria was almost equal to the activity of rotenone-insensitive NADH-cytochrome c reductase. 3. Mitochondrial LDH acting as an oxidase of lactate to pyruvate may feed an "external" pathway, but the activity of the mitochondrial enzyme is a limiting factor in oxidation of lactate-derived NADH. 4. Mitochondrial LDH acting as a reductase of pyruvate to lactate successfully competes with an "external" pathway for cytoplasmic NADH. 5. Exogenous NADH oxidation via an "external" pathway was inhibited by pyruvic acid. This inhibition was overcome by addition of oxamic acid or hydrazine.     

Significance of bacterial ectoenzymes in aquatic environments

The report presents studies on temporal and spatial variations of kinetics (Vmax and Km) of bacterial ectoenzyme activity (-glucosidase - Glc, leucine aminopeptidase - Leu-amp) in the naturally eutrophic Plusee. Glc and Leu-amp activity were positively correlated with the flux of polymeric materials (polysaccharides, proteins) in the lake. Glc activity was low when algal populations grew actively, but during the algal bloom breakdown Glc activity increased rapidly. Leu-amp displayed the highest rates of activity in the epilimnion and was tightly coupled to bacterial production. The synthesis of studied ectoenzymes was under control of a repression/derepression mechanism. The significance of ectoenzymes for the transformation and bacterial utilization of organic matter, and their role in the microbial loop in aquatic environments is discussed.