The use of actimetry to assess changes to the rest-activity cycle

The endogenous circadian oscillator (the body clock) is slow to adjust to altered rest-activity patterns. As a result, several negative consequences arise during night work and after time-zone transitions. The process of adjustment can be assessed by measurements of the sleep electroencephalogram (EEG), core temperature or melatonin secretion, for example, but these techniques are very difficult to apply in field studies, and make very great demands upon both experimenters and subjects. We have sought to establish if the activity record, measured conveniently and unobtrusively by a monitor attached to the wrist, can be treated in ways that enable estimates to be made of the disruption caused by changes to the rest-activity cycle, and the process of adjustment to them. In Part A, we describe the calculation and assessment of a series of “activity indices” that measure the overall activity pattern, activity when out of bed or in bed, or the activity in the hours adjacent to going to bed or getting up. The value of the indices was assessed by measuring changes to them in subjects undergoing night work or undergoing time-zone transitions. In both cases, there is a large body of literature describing the changes that would be expected. First, night workers (working 2 to 4 successive night shifts) were investigated during rest days and night shifts. The indices indicated that night work was associated with lower activity when the subjects were out of bed and higher activity when in bed. Some indices also measured when subjects took an afternoon nap before starting a series of night shifts and gave information about the process of adjustment to night work and recovery from it. Second, in studies from travelers crossing six or more time zones to the east or west, the indices indicated that there were changes to the rest-activity cycle immediately after the flights, both in its overall profile and when activity of the subjects in bed or out of bed was considered, and that adjustment took place on subsequent days. By focusing on those indices describing the activity records during the last hour in bed (LHIB) and the first hour out of bed (FHOB), some evidence was found for incomplete adjustment of the body clock, and for differences between westward and eastward flights. In Part B, the battery of indices are applied to the activity records of long-haul pilots, whose activity patterns showed a mixture of effects due to night work and time-zone transitions. Actimetry was performed during the flights themselves and during the layover days (which were either rest or work days). The indices indicated that all pilots had disrupted rest-activity cycles caused by night flights, and that there were added problems for those who had also undergone time-zone transitions. Rest days were valuable for normalizing the activity profile. For those pilots who flew to the west, adjustment was by delay, though not all aspects of the rest-activity cycle adjusted immediately; for those who flew to the east, some attempted to advance their rest-activity cycle while others maintained home-based activity profiles. The indices indicated that the activity profile was disrupted more in those pilots who attempted to advance their rest-activity cycle. We conclude that objective estimates of the disruption caused to the rest-activity cycle and the circadian system can be obtained by suitable analysis of the activity record.     

Application of spectrophotometric methods to the determination of protein bound to agarose beads.

The proteolytic activities of α-chymotrypsin, trypsin, pepsin, bromelain, and an extract from germinating pumpkin seeds were determined by their ability to effect the release of 1-anilino-8-naphthalenesulfonate bound to internal hydrophobic sites in intact protein substrates resulting in a decline in fluorescence. Casein, glyceraldehyde-3-P dehydrogenase, urease, catalase, pumpkin seed globulin, and bovine serum albumin enhanced the fluorescence of 1-anilino-8-naphthalenesulfonate sufficiently to be used as proteolytic substrates in this assay procedure. The activity of 1 μg chymotrypsin or trypsin and 100 ng pepsin could be easily detected by this method within 4 to 8 min depending upon the protein substrate. The digestive enzymes and bromelain exhibited activity against most if not all six of the protein substrates used. In contrast, the extract from germinating pumpkin seeds exhibited significant activity only against pumpkin seed globulin, with maximal activity at pH 7.4. Compared with the assay method for proteolytic activity utilizing ninhydrin analysis of the reaction products, this method was at least 10 times more rapid and gave significant detectable activity with much lower quantities of proteolytic enzyme.     

Transport of yeast vacuolar trehalase to the vacuole

We have tested yeast secretory mutants, which define different stages of the secretory pathway, for their levels of vacuolar trehalase activity. Mutations that cause accumulation of secretory proteins in the endoplasmic reticulum or in the Golgi body lead to diminished vacuolar trehalase activity. Mutations that cause accumulation of secretory vesicles have no effect on vacuolar trehalase activity. None of the mutations affects cytoplasmic trehalase activity. These results provide further evidence for the existence of a compartmentalized trehalase in yeast, and demonstrate that the enzyme enters the secretory pathway.     

Contribution of cardiopulmonary baroreceptors to the control of the kidney.

The role of cardiopulmonary receptors in the control of renal sympathetic nerve activity and of renin release is reviewed. The evidence indicates that cardiopulmonary receptors with vagal afferents exert a tonic inhibition on both renal nerve activity and on renin release. The magnitude of this inhibition appears directly related to changes in blood volume. Atrial as well as ventricular receptors can influence the secretion of renin. Cardiopulmonary receptors with vagal afferents may also reflexly modulate renal prostaglandin secretion. There is preliminary evidence to suggest that cardiopulmonary receptors with sympathetic afferents can influence renal nerve activity. The limitations of previous studies are outlined and a direction for future studies is suggested. It is concluded that alterations in cardiopulmonary vagal afferent input and the resulting changes in renal nerve activity and in renin release are appropriate for the maintenance of blood volume homeostasis.     

Strain differences of the ability to hydroxylate methotrexate in rats

Converting activity of methotrexate (MTX) to 7-hydroxymethotrexate (7-OH-MTX) was examined using eight strains of rats. Marked variability of the activity was found in liver cytosols from the rats. The highest activity was observed with Sea:SD rats, followed by LEW/Sea and Jcl:Wistar rats. The lowest activity was observed with WKA/Sea rats. The difference in the activity between Sea:SD and WKA/Sea strains was 104-fold. The variation was correlated to the strain difference of benzaldehyde oxidase activity in the rats. The cytosolic 7-hydroxylase activities in other tissues of Sea:SD rats were much higher than those of WKA/Sea, similarly to the case in liver. The liver microsomes of Sea:SD rats exhibited no 7-hydroxylase activity toward MTX even in the presence of NADPH. The cytosolic 7-hydroxylating activity of the livers of Sea:SD rats was inhibited by menadione, β-estradiol, chlorpromazine and disulfiram, inhibitors of aldehyde oxidase, but not oxypurinol, an inhibitor of xanthine oxidase. The purified aldehyde oxidase from the livers of Sea:SD rats exhibited a significant 7-hydroxylating activity toward MTX. However, xanthine oxidase had no ability to hydroxylate MTX. These facts suggest that MTX hydroxylating activity in rats is predominantly due to aldehyde oxidase, and the strain differences are due to the variations of the flavoenzyme level.     

Effect of N-methyl-D-aspartate applied to the ventral surface of the medulla on the trachea

Structures located near the ventral surface of the medulla (VMS) affect both cardiovascular tone and respiratory activity. In addition cooling the intermediate area of the VMS blocks the increases in parasympathetic activity and tracheal tone resulting from ventilation with hypercapnic or hypoxic gas mixtures, or due to stimulation of mechanoreceptors within the lung. Since cooling the surface of the VMS may affect fibers of passage as well as cell bodies, we performed studies in which pledgets containing N-methyl-D-aspartic acid (NMDA), a synthetic excitatory amino acid, were applied to intermediate area of the VMS. The studies were performed in chloralose-anesthetized, artificially ventilated cats. Application of pledgets containing NMDA (10(-7) mol at 10(-3) M) caused increases in tracheal pressure and the onset of phasic phrenic activity, but application of 10(-8) mol at 10(-4) M of NMDA could produce tracheal constriction without the appearance of phasic phrenic activity. Applying to the entire VMS either 2-amino-5-phosphonovalerate (2-APV, 10(-6) M), a specific antagonist to NMDA, or lidocaine (2%), a local anesthetic, 60 s before the application of pledgets containing NMDA, prevented the increase in tracheal tone and phasic phrenic activity. Intravenous administration of atropine methyl nitrate 0.5 mg/kg, a cholinergic antagonist, blocked tracheal responses to local application of pledgets containing NMDA but did not affect the increase in phasic phrenic nerve activity. These findings suggest that when stimulated, neurons near the surface of the VMS in the vicinity of the intermediate area increase the activity of parasympathetic fibers to the airway.     

Concanavalin A-induced translocation of part of the GTP-binding activity from the membrane to the cytosol in murine thymocytes

Concanavalin A (Con A) stimulation resulted in the rapid redistribution of part of the GTP-binding activity from the membrane to the cytosol in murine thymocytes. This change in GTP-binding activity was dependent on the Con A concentration. To investigate the relationship between this redistribution and phospholipase C (PLC) activity, the effect of GTP gamma S on the cytosol PLC activity was also examined, and it was found that GTP gamma S enhanced the phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis activity in the cytosol of Con A-stimulated thymocytes more than in that of unstimulated thymocytes. This enhancement by GTP gamma S was also dependent on the Con A concentration. The results suggest that in murine thymocytes, the GTP-binding protein (G-protein) involved in the regulation of PLC activity may be translocated from the membrane to the cytosol upon Con A stimulation. Besides, the dose dependence curve for the change in the GTP gamma S-binding activity was similar to that for inositol phosphates formation in Con A-stimulated thymocytes, suggesting that the translocation of the G-protein is closely related to PLC activation. Furthermore, the effects of cytosol fractions containing the 38-43 and 23-28 kDa GTP-binding subunits of G-proteins on the PIP2 hydrolysis activity of partially purified PLC were examined. The fraction containing the 23-28 kDa subunit evidently enhanced the PLC activity but that containing the 38-43 kDa subunit enhanced the activity to a much lower extent. Moreover, the 23-28 kDa subunit fraction of Con A-stimulated thymocytes was more effective as to enhancement of the PLC activity than that of unstimulated thymocytes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Day-night differences in the response of the pineal gland to swimming stress

The effect of swimming stress on pineal N-acetyltransferase activity, hydroxyindole-O-methyltransferase (HIOMT) activity, and melatonin content was studied during the day and night in adult male rats. At night, elevated pineal activity was suppressed by light exposure before the animals swam. During the day, swimming for 2 hr did not stimulate NAT activity unless the animals were pretreated with desmethylimipramine (DMI), a norepinephrine uptake blocker. Pineal melatonin content after daytime swimming exhibited a weak rise, unless DMI was injected, in which case melatonin levels showed a highly significant increase. Swimming at night caused a greater (compared to daytime levels) increase in NAT activity in both noninjected and DMI-injected rats. Melatonin levels at night were highly significantly stimulated (compared to daytime values) even without pretreatment of the rats with DMI. The greater response of the rat pineal to swimming stress at night may relate either to an increase in the number of beta-adrenergic receptors in the pinealocyte membrane at night or to a reduced capacity of the sympathetic neurons in the pineal to take up excess circulating catecholamines. Pineal HIOMT activity was not influenced by swimming (with or without DMI) either during the day or at night.     

Peroxisomal participation in the cellular response to the oxidative stress of endotoxin

Exposure to a sublethal dose of endotoxin offers protection against subsequent oxidative stresses. The cellular mechanisms involved in generating this effect are not well understood. We evaluated the effect of endotoxin on antioxidant enzymes in liver peroxisomes. Peroxisomes have recently been shown to contain superoxide dismutase (SOD) and glutathione peroxidase (GPX) in addition to catalase. Peroxisomes were isolated from liver homogenates by differential and density gradient centrifugations. Endotoxin treatment increased the specific activity of SOD and GPX in peroxisomes to 208% and 175% of control activity, respectively. These findings correlated with increases in peroxisomal SOD and GPX proteins observed by immunoblot. Although the quantity of catalase protein was increased when assessed by immunoblot analysis, the specific activity of catalase was decreased to 68% of control activity. Activation of catalase with ethanol only restored catalase activity to control levels suggesting that catalase had undergone irreversible inactivation. The observed increase in GPX activity may represent a compensatory mechanism triggered by accumulating H₂O₂. The data presented here suggest for the first time that mammalian peroxisomal antioxidant enzymes are altered during the oxidative injury of endotoxin treatment.     

Iron--dioxygen-dependent changes to the biological activities of bleomycin

Antitumor antibiotic bleomycin can bind to and degrade DNA, both in vivo and in vitro. This DNA damaging property in vitro can be related to its ability to chelate ferrous ions under aerobic conditions leading to the formation of "active oxygens," which are thought to be directly responsible for the damage. At present, evidence points to the hydroxyl radical formed by an iron-catalyzed Haber-Weiss reaction as the free radical most likely to be involved in this damage. When these same reactions occur in the absence of DNA, the free radicals then damage the bleomycin molecule, resulting in changes to its DNA-degrading activity, antibacterial properties, and chemical composition. Attempts to protect both bleomycin and DNA with a variety of specific and nonspecific scavengers have been unsuccessful, with several even showing pro-oxidant activity towards the iron-dependent damage. Only the metal chelators were effective inhibitors of bleomycin-iron-dependent damage to DNA. The damaged bleomycin lost some 50% of its ability to degrade DNA in vitro. This activity was closely paralleled by a loss in antibacterial activity against two different strains of bacteria.     

The effect of adaptation to stress exposures and to periodic hypoxia on the cardiomyocyte trigger activity of the papillary muscles in the rat]

Action potential of cardiomyocytes was recorded in experiments on isolated papillary muscle of the rat left ventricle. The effect was estimated of preliminary adaptation to intermittent hypobaric hypoxia or to short-term stress exposure on the incidence and the pronouncement of delayed after depolarization and of trigger activity induced by a high frequency stimulation against the background of isoproterenol (10(-8) M). It was shown that adaptation to hypoxia or to stress reduced the incidence of delayed after depolarization and of trigger activity, adaptation to stress exerting a more pronounced effect. Immobilization stress (6 hours) potentiated the trigger activity, adaptation to stress exerting a more pronounced effect. Immobilization stress (6 hours) potentiated the trigger activity and this potentiation was effectively prevented by either type of adaptation.     

Releasing of plasminogen-activator from the kidney to the blood (author's transl)]

When fibrinolytic activity in blood samples from various vessels was examined by the dilute-blood-clotlysis-time method (DBCLT), it was found to be noticeably high in the renal venous blood, though the activity was not detected by usual blood clotlysis time method. Plasmin was not detected in any blood samples examined, and the contents of fibrinogen and fibrin (or fibrinogen) breakdown products in the renal venous blood were not significantly different from those in the blood from other vessels. However, the high activity of plasminogen-activator was found only in the renal venous blood. Inhibitors on plasmin and plasminogen-activator (urokinase) were detected in almost the same amount in the blood samples from the various vessels. The amount of the inhibitors was sufficient to inhibit the plasminogen activation by urokinase, whose activity was equivalent to the plasminogen-activator activity in the renal venous blood. These results indicate that the high activity by DBCLT in the renal venous blood was derived from the high activity of plasminogen-activator, which was inactivated by inhibitors in undiluted blood. Plasminogen-activator may be released from the kidney to the blood, and immediately inactivated by the inhibitors in renal vein, and then diluted with systemic blood which contains little plasminogen-activator.     

Differences in the activity pattern of the wild boar Sus scrofa related to human disturbance

Over the last century, human activity has caused significant changes to the activity patterns of many wildlife species. The wild boar is one species known to change its activity pattern with the intensity of human disturbance. We conducted camera trap surveys in two study sites, Shingo and Himuro, in Tochigi, central Japan. We investigated effects of two types of human disturbance on the activity pattern of a wild boar population: ‘direct’ disturbance related to hunting activity and ‘indirect’ disturbance related to daily human activity. In the hunting season, relative abundance indices (RAI) of wild boars significantly decreased, and the proportion of activity at night increased compared with the nonhunting season. RAI of wild boars at night decreased with increasing distance from the settlement, while RAI of wild boars during the day did not. Relative proportion of activity at night was higher in cameras at 0–200 m from the settlements, while no significant pattern was found in cameras far from settlements. Both direct and indirect effects of human activity had a significant effect on the activity pattern of wild boars. A decrease in human activity may result in the rapid expansion of wild boar populations, and re-evaluation of the human factor is important for more intelligent management of wild boar populations and to solve the human–wildlife conflict.     

Cyclic activity of the receptors of cobalamin bound to transcobalamin II

The activity of receptors specific for human transcobalamin II-Cobalamin (TC II-Cbl) were measured in virus-transformed lymphoblasts, hepatocytes (hepatoma) and diploid fibro lasts. In all three types of human cells the receptor activity increased as cells went from a resting phase to the most actively dividing phase. Receptor activity declined as cell division slowed. The changes in activity of lymphoblasts and hepatocytes were produced by changes in receptor number and not by changes in affinity between receptors and TC II-Cbl. The basis of the change in fibroblasts was not clear. The Cbl-dependent methionine synthetase activity of fibroblasts, in contrast, tended to be greatest when the cultures were confluent and replication had slowed. As the fibroblasts became senescent the receptor activity for TC II-Cbl declined and the fluctuations with the phase of the cell were blunted. However, the release of apo TC II from the cells was maintained. These observations must be taken into consideration when the respective cells are used as models. Even more important are the implications of the observations of the changes in receptor activity for TC II-Cbl for the regulation of the entry of Cbl into cells.     

The dynamics of neuron activity level in the striatum of the monkey brain in relation to the realization of multistage behavior

Level of the unit activity was studied in the monkey putamen during multistage behavior. Two groups of neuron activity patterns were distinguished. The first one involved patterns of low level neuron activity less exceeding the background level than the second one; the other group involved patterns of high level neuron activity exceeding the background level in the second time. These kinds of patterns were behavior-related. Patterns with low level neuron activity were recorded preferentially in relation to the trigger stimuli and reward. Patterns of high level neuron activity were recorded in relation to the decision-making, movements of arms in the left and right directions. Besides, their number rose in relation to the auditory cue reported to right realization of the task. It was established that the number of patterns of high level neuron activity rose in key moments of behavior, while the number of the patterns of low level neuron activity decreased.     

Use of stable isotopes to measure the metabolic activity of the human intestinal microbiota

The human intestinal microbiota is a complex biological system comprising a vast repertoire of microbes with considerable metabolic activity relevant to both bacterial growth and host health. Greater strides have been made in the analysis of microbial diversity than in the measurement of functional activity, particularly in vivo. Stable isotope probing offers a new approach by coupling measurements of metabolic activity with microbial identification. Using a low-enrichment labeling strategy in vitro, this study has identified metabolically active bacterial groups via magnetic-bead capture methodology and stable isotope ratio analysis. Using five probes (EUB338, Bac303, Bif164, EREC482, and Clep866), changes in the activities of key intestinal microbial groups were successfully measured by exploiting tracers of de novo RNA synthesis. Perturbation of the nutrient source with oligofructose generated changes in the activity of bifidobacteria as expected, but also in the Bacteroides-Prevotella group, the Eubacterium rectale-Clostridium coccoides group, and the Clostridium leptum subgroup. Changes in activity were also observed in response to the medium type. This study suggests that changes in the functional activity of the gut microbiota can be assessed using tracers of de novo nucleic acid synthesis combined with measurement of low isotopic enrichment in 16S rRNA. Such tracers potentially limit substrate bias because they are universally available to bacteria. This low-enrichment labeling approach does not depend on the commercial availability of specific labeled substrates and can be easily translated to in vivo probing experiments of the functional activity of the microbiota in the human gut.     

Characteristics of the immunocyte cooperation in 2 strains of mice opposite in their sensitivity to dermaphytoses during the immune response to antigens of the causative agent

C57BL/6 and BALB/c mice, opposite in their sensitivity to dermatophytic infections, show similar activity of phagocytes as regards their capacity for the destruction of injected conidia of dermatophytes, but differ in the changes of this activity after immunization with dermatophytic antigenic complexes (DAC). Shortly after the injection of the antigens, the lymphocyte-mediated suppression of the fungicidal activity of macrophages, caused by the interaction of DAC with intact T-lymphocytes, was detected in the animals of both strains. Later in C57BL/6 mice resistant to mycosis the formation of cell-mediated immunity to DAC occurs, with the simultaneous production of factor stimulating the fungicidal activity of macrophages. In BALB/c mice sensitive to mycosis the injection of DAC induces active antibody production, but not the formation of delayed hypersensitivity with the resulting stimulation of the fungicidal activity of phagocytes. The injection of DAC into mice of the above-mentioned strains induces changes, peculiar to each strain, in the mitogen-induced proliferation of spleen cells and in the character of immune response to sheep red blood cells. Differences in the influence of DAC on the induction of immune response in C57BL/6 and BALB/c mice are realized by cells belonging to the population of T-lymphocytes.     

Inducible Formation of Glutamate Dehydrogenase in Rice Plant Roots by the Addition of Ammonia to the Media

The activity of glutamate dehydrogenase (l-glutamate: NAD oxidoreductase, EC 1.4.1.2.; GDH) of rice plants changes in response to the nitrogen source supplied to the culture solution. The activity of NADH-GDH(aminating) in roots is rapidly increased by the addition of ammonia, whereas the activity in shoots is much less affected by nitrogen supply. The activity increased with increasing concentration of ammonia at least up to 14.3 mM. In roots GDH activity was found in both the mitochondrial and soluble fractions. The increase of NADH-GDH activity caused by the ammonia treatment occurs mainly in the latter fraction. The new band with GDH activity was detected on the zymogram of polyacrylamide gel electrophoresis and this inducible enzyme is active with both NAD and NADP. On the other hand, the constitutive enzyme activity active with NAD is also increased by the ammonia treatment. The increase of enzyme activity is prevented by the addition of cycloheximide or chloramphenicol to culture medium. The incorporation of ¹⁴C-leucine(U) into GDH proteins was also studied using polyacrylamide gel electrophoresis. Higher radioactivity was found in induced samples than in non-induced ones. These results show that the increase of GDH activity in roots by ammonia treatment seems to depend on de novo protein synthesis.     

Adaptation of intestinal disaccharidases to saccharose in the ontogeny of chicks

In experiments of 1, 15 and 30-day chicks, studies have been made of adaptational changes in the activity of maltase and saccharase from different parts of the small intestine during feeding by sucrose. It was found that the increase in the activity of the mentioned enzymes during sucrose utilization takes place only in 30-day chicks. At earlier stages of ontogenesis, adaptational changes in the activity of disaccharidases are directed to the enhancement of the decrease in the activity of maltase and saccharase in the small intestine, this decrease being observed at these stages in control chicks.     

Differential effect of N-carboxymethylisatoylation on the DNA polymerase activity, the 5' leads to 3'-exonuclease activity and the 3' leads to 5'-exonuclease activity of DNA polymerase I of Escherichia coli

Treatment with native DNA polymerase I of Escherichia coli with the acylating agent N-carboxymethylisatoic acid anhydride (NCMIA) results under specific conditions in a rapid loss of polymerase activity, an increase in 5' leads to 3'-exonuclease activity and in unchanged 3' leads to 5'-exonuclease activity. When a nucleoside triphosphate and Mg2+ was present the polymerase activity was completely protected against the effect of NCMIA. Treatment with higher concentration of the acylating agent under these conditions led to a loss of 3' leads to 5'-exonuclease activity without any appreciable loss of polymerase activity. Treatment with NCMIA of the two catalytically active fragments of the enzyme led to very similar results. In this case both the polymerase activity and the 3' leads to 5'-exonuclease activity deteriorated more rapidly on treatment with the acylating reagent. The increase in 5' leads to 3'-exonuclease activity as a result of modification of the native enzyme appeared to be due to a change in the optimum conditions with regard to concentration of the assay buffer used. These changes are very similar to those seen when the polymerase is cleaved by limited proteolysis. From the results obtained it is concluded that NCMIA reacts primarily with a site at or near the triphosphate-Mg2+ complex binding site, leading to an almost complete loss of polymerase activity. The acylating reagent reacts also with another group on the native enzyme resulting in a modification of the 5' leads to 3'-exonuclease activity, and at high concentrations with a group leading to a slow loss of 3' leads to 5'-exonuclease activity.