LD Activity Research Articles

Abstract We114: Feeding Behavior Underlies the Circadian Rhythm in the Autonomic Input to the Heart and Heart Rate

Objective: The circadian pacemaker located in the suprachiasmatic nucleus (SCN) of the hypothalamus drives circadian rhythms in feeding and activity. Several studies suggest the SCN regulates autonomic input to the heart to generate day-night rhythms in heart rate. Hypothesis: The circadian rhythm in feeding generates day-night rhythms in autonomic input to the heart and heart rate. Methodology: Mice (n=6/sex) were implanted with telemetry probes to continuously record heart rate, core body temperature, and activity in thermoneutral housing conditions. Mice were housed in a 12 h light: 12 h dark cycle (LD) with ad libitum access to food (ALF). In these conditions, mice primarily eat during the dark cycle. Mice were subjected to light phase time-restricted feeding (TRF) for 5 days. This forces the mice to eat during the light cycle. We quantified changes in autonomic input to the heart by injecting mice with the autonomic receptor inhibitors propranolol and methylatropine. To determine if circadian rhythms generate day-night rhythms in autonomic input to the heart, TRF mice were switched from LD to constant darkness (DD) and then returned to ALF in DD. Results: ALF mice housed in LD had 24-hour rhythms in autonomic input to the heart, heart rate, and activity that all peaked in alignment during the dark cycle. One day after starting TRF, the phase of the 24-hour rhythms in heart rate but not activity shifted by 8-10 hours to align with feeding during the light phase. Switching TRF mice in LD to DD did not alter the phase or amplitude of the day-night rhythms in autonomic input to the heart, heart rate, or activity. Returning the TRF mice in DD to ALF showed the autonomic input to the heart and heart rate re-aligned with the free running rhythm in activity. Conclusions: Day-night rhythms in heart rate and autonomic input to the heart align with activity in LD and DD in ad libitum fed but not time-restricted fed conditions. These data demonstrate that feeding behavior, not SCN-signaling or activity rhythms, drives day-night rhythms in autonomic input to the heart and heart rate.

ANTIOXIDANT ACTIVITY EVALUATION OF KALANCHOE PINNATA (LAM.) PERS. FRESH LEAF JUICE

Objective: The objective of this study was to determine the antioxidant and free radical scavenging properties of Kalanchoe pinnata (Lam.) Pers. fresh leaf juice for standardized doses using different types of assays. Methods: The investigation of the antioxidant activity was carried out for the previously standardized doses (by cytotoxicity and genotoxicity analysis) – 50 μl (low dose - LD) and 70 μl (high dose-HD) by Phosphomolybdenum (PM), Ferric Reducing Antioxidant Power (FRAP), 2, 2-diphenyl-1- picrylhydrazyl (DPPH) radical scavenging activity, and 2, 2’-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging activity assays. Results: The activity measured by PM assay for LD and HD of the extract was equivalent to 109 μg/ml and 141 μg/ml of ascorbic acid, respectively, while by FRAP assay, it was equivalent to 17.5 μg/ml and 47 μg/ml of ascorbic acid, respectively. The activity measured by DPPH assay was found equivalent to 83 μg/ml and 177.5 μg/ml of ascorbic acid for LD and HD, with the percent scavenging activity 4.55% and 17.04%, respectively. Similarly, the activity of LD and HD by ABTS assay was found equivalent to 277.5 μg/ml and 308 μg/ml of ascorbic acid with the percent scavenging activity 39.01% and 47.93%, respectively. Conclusion: The changes observed in the absorbance values for all the assays reflected the increasing antioxidant activity of the plant extract in dose-dependent manner, which justifies the traditional use of this herb in prevention of diseases induced by oxidative stress. The antioxidant activity of K. pinnata fresh leaf juice has been conducted for the 1st time and has not been previously reported.

Circadian Rhythm of Nitrate Reductase Activity in Jatropha curcas Under Different Photoregimens

Our knowledge on the circadian phenomenon in Jatropha curcas is limited. Therefore, we studied the activity of nitrate reductase (NR) in leaves of J. curcas under light and dark cycles (LD 12:12), continuous light (LL) and constant darkness (DD). We observed a statistically significant circadian rhythm in NR activity in LD. Unlike in DD, the circadian oscillation in NR activity persisted in continuous light, irrespective of different light intensities (LL 2 lx and 15 lx). The peak activity of NR appeared in the morning in LL 15 lx and forenoon in LD, while it reached a peak at night in LL 2 lx. This shows that rhythm in NR activity in J. curcas could be endogenous and that the light intensity might modulate acrophase of NR activity. Jatropha curcas could be considered as an ideal higher plant model for teaching NR activity rhythm to undergraduate and postgraduate students of biology streams.

Leadership development in Higher Education: A literature review and implications for programme redesign

AbstractLeadership development (LD) activity and its effectiveness has not been explored rigorously across changing university settings globally. As Higher Education settings change radically throughout the world, Higher Education professionals are operating in more uncertain environments, and leaders are taking increasingly complex and diverse approaches to their leadership roles. LD activities therefore become important in supporting this highly complex context, yet little is known in the literature about LD and its impact in Higher Education. We examine peer‐reviewed work on LD in Higher Education settings globally to understand what may be learned about its content, processes, outcomes and impact. Our results suggest the current literature is small‐scale, fragmented and often theoretically weak, with many different and coexisting models, approaches and methods, and little consensus on what may be suitable and effective in the Higher Education context. We reflect on this state of play and develop a novel theoretical approach for designing LD activity in Higher Education institutions.

Hyperoside protects human kidney‑2 cells against oxidative damage induced by oxalic acid.

The majority of renal calculi (kidney stones) are calcium stones. Oxidative damage to renal tubular epithelial cells induced by reactive oxygen species (ROS) is the predominant cause of calcium oxalate stone formation. Hyperoside (Hyp) is a flavonol glycoside extracted from medicinal plants and appears to exhibit potent antioxidant activity in various cells. The aim of the present study was to investigate the protective effect of Hyp on renal cells exposed to oxidative stress simulated by oxalic acid (OA), and to determine whether the underlying mechanism involves the nuclear factor E2‑related factor2 (Nrf2)‑antioxidative response element signaling pathway. The study determined the indicators of high oxidative stress, including ROS and hydrogen peroxide (H2O2) in human kidney‑2 cells and the results demonstrated that the levels of ROS, as evaluated by flow cytometry, and H2O2 were significantly increased following treatment with OA (5mmol/l) for 24h (OA group), compared with those in the untreated control group. The increased activity of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in these cells explained this observation, as it is a major source of ROS. The results demonstrated that, in the OA group, the adhesion of calcium oxalate crystals and lactate dehydrogenase (LDH) were significantly increased, and MTT assay demonstrated that cell viability was inhibited, compared with the control, which indicated that severe injury of cells was induced by OA. However, when the cells were pre‑treated with Hyp prior to treatment with OA (drug group), the levels of ROS and H2O2, and the activities of NADPH oxidase and LD were increased, and the adhesion of calcium oxalate crystals to cells was reduced, compared with the OA group. Western blot analysis and reverse transcription‑quantitative polymerase chain reaction demonstrated that the protein and mRNA expression levels of Nrf2, heme oxygenase‑1 (HO‑1) and NAD(P)H: quinineoxidoreductase 1 (NQO1) in the Hyp groups were significantly increased, compared with those in the OA group, with the exception of Nrf2 mRNA. These results suggested that Hyp had a marked protective effect on renal cells against the oxidative damage and cytotoxicity simulated by OA. This is the first report, to the best of our knowledge, demonstrating that the ability of Hyp to enhance the endogenous functions of antioxidation and detoxification in cells may involve the Nrf2/HO‑1/NQO1 pathway.

The Use of Software Tools in Linked Data Publication and Consumption

To reduce the complexity intrinsic to LD manipulation, software tools are used to publish or consume data associated to LD activities. However, few developers have a broad understanding of how software tools may be used in publication or consumption of Linked Data. The goal of this work is to investigate the use of software tools in Linked Data publication and consumption processes. More specifically, understanding how these software tools are related to process of publication or consumption of LD. In order to meet their goal, the authors conducted a Systematic Literature Review (SLR) to identify the studies on the use of software tools in these processes. The SLR gathered 6473 studies, of which only 80 studies remained for final analysis (1.25% of the original sample). The highlights of the study are: (1) initial steps of the publication process are fairly supported by the software tools; (2) Non-RDF serialization is fairly supported in publication and consumptions process by the software tools; and (3) there are non-supported steps in consumption and publication processes by the tools.

Effect of 2-deoxy-d-glucose induced metabolic stress on testicular steroidogenesis and antioxidant status in golden hamster,Mesocricetus auratus: Role of photoperiod.

The driving force underlying the evolution of reproductive seasonality is the marked seasonal fluctuation in energy availability. It is well established that photoperiod regulates testicular steroidogenesis through modulation of circulatory and local melatonin levels and MT1R expression in golden hamster. However, photoperiodic variation in energy availability and its impact on testicular steroidogenesis and antioxidant status has never been investigated for any seasonal breeder. Therefore, the aim of the present study is to elucidate the role of photoperiod in modulation of metabolic stress induced variation in steroidogenesis and antioxidant status in testes. We experimentally reduced energy availability via administration of 2-deoxy-d-glucose (2-DG), a synthetic glucose analog, in critical- (CP), long- (LD) and short-day (SD) exposed golden hamsters, Mesocricetus auratus and examined testicular steroidogenesis and antioxidant status. Administration of 2-DG decreased testicular steroidogenesis and antioxidant enzyme activity in CP and LD experienced hamsters as compared to CP- and LD-control groups. Moreover, a decrease in the plasma- and local-melatonin level and MT1R expression in testes with a simultaneous increase in plasma corticosterone level was noted following 2-DG administration in CP and LD exposed hamsters. In contrast, no significant effect of 2-DG administration was observed on the parameters investigated under SD condition. Therefore, it can be suggested that the reduction in energy availability under CP and LD condition suppressed testicular steroidogenesis and increased oxidative damage in testis whereas SD induced melatonin might have buffered the 2-DG induced suppression of testicular steroidogenesis and maintained testicular antioxidant status.

The effect of different systems of carrying backpacks on muscle activity

The purpose of this study is to compare the muscles activity in different systems of carrying the load in students and mountain climbers. EMG activity for TA and LD muscles were recorded with surface electrode while walking with different conditions. 26 young students attended three test sections in three days apart. After warm up subjects walked 10 min on the treadmill without backpack as a normal position in the first day. On the second day subjects walked 10 min with normal backpack, and on third day they walked 10 min with counterbalance load backpack. EMG activity of the TA and LD measured during three conditions of walking. One way analysis of variance (ANOVA) was applied on EMG patterns at muscles. The results indicated that there is a significant difference in TA and LD activities after walking with normal backpack and walking without backpack. There is an also significant difference in TA and LD activities between walking with normal backpack and counterbalance backpack. Results also indicated that there is no significant difference between walking without backpack and walking with counterbalance backpack with p<.856. Findings of this study clearly show the advantage of a counterbalance system for carrying the loads. In addition to these, the reduction of muscle activity is a considerable kinematic and ergonomic benefit of carrying load in such backpacks.

Lipid nanocarriers containing a levodopa prodrug with potential antiparkinsonian activity.

This paper describes the production, characterization and in vivo activity of lipid nanocarriers (LN) containing a levodopa prodrug (LD-PD) with therapeutic potential in Parkinson's disease. LD is the mainstay of the pharmacotherapy of Parkinson's disease. However, after a good initial response, motor fluctuations, dyskinesia and loss of efficacy, develop over time, partly due to oscillations in plasma and brain levels of the drug. LD-PD was produced with the aim of prolonging the pharmacological activity of LD. To improve solubility, and simultaneously provide a long lasting release and therapeutic efficacy, the prodrug was formulated in tristearin/lecithin LN. The obtained formulation was homogeneous in particle size and remained stable for up to 2months from preparation. For the three different tested LD concentrations, namely 1.25, 2.5 and 5.0mg/ml, the morphological characterization revealed no substantial differences between unloaded and LD-PD loaded LN. The calorimetric test showed an interaction between the lipid phase and the loaded prodrug. In vitro studies using the dialysis method and enzymatic degradation procedure showed that the LD-PD loaded LN provided a controlled prodrug release. Finally, two behavioural tests specific to akinesia (bar test) or akinesia/bradykinesia (drag test) performed in 6-hydroxydopamine hemilesioned mice (a model of Parkinson's disease) demonstrated that the LD-PD loaded LN attenuated parkinsonian disabilities, showing a slightly reduced maximal efficacy but a longer lasting action (up to 24h) than an equal dose of LD. We conclude that LD-PD loaded LN may represent a future LD formulation useful in Parkinson's disease therapy.

Encapsulation of &lt;I&gt;Trans&lt;/I&gt;-Dehydrocrotonin in Liposomes: An Enhancement of the Antitumor Activity

The aim of this study was the encapsulation of trans-dehydrocrotonin (t-DCTN) and its inclusion complexes with hydropropyl-beta-cyclodextrin (HP-beta-CD) in liposomes to improve t-DCTN antitumor activity. The in vitro kinetic profiles of t-DCTN-loaded liposomes (LD) and t-DCTN:HP-beta-CD-loaded liposomes (LC) were evaluated using the dialysis technique. The antitumor activity of LD and LC were investigated against Sarcoma 180 in Swiss mice. Histopathological and hematological analyses were carried out. The amounts of t-DCTN and t-DCTN:HP-beta-CD inclusion complex encapsulated in liposomes were equivalent to 1 mg of t-DCTN. The encapsulation efficiencies of LD and LC were 95.0 +/- 3.8% and 91.1 +/- 5.6%, respectively. In relation to kinetics, the drug release profiles of t-DCTN are in substantial agreement with the Fickian model. The treatment of animals with LD and LC produced tumor inhibitions of 79.4 +/- 9.6% and 63.5 +/- 5.5%, respectively. The liposomal encapsulation of t-DCTN by entrapment in the phospholipid bilayer increased at twice the antitumor activity. Moreover, the liposomal formulations reduced the hepatotoxicity effect of the drug and no significant hematological toxicity was observed in the treated animals. However, the counting of platelets was slightly decreased. Thus, the results show that the development of liposomal formulations containing t-DCTN or t-DCTN:HP-beta-CD is an important advance for enabling this drug to be use in therapy.

Effects of Photoperiod on Alternative Respiration Pathway in Nectarine Flower Buds During Dormancy Induction

Abstract Characteristics of dormancy induction and alternative respiration pathway (also known as cyanide-resistant respiration) of nectarine flower buds in different photoperiods were studied to determine the function of photoperiod and alternative respiration pathway in dormancy induction. Oxygen-electrode system and respiratory inhibitors were used to measure total respiratory rates and rates of alternative respiration pathway. The results showed that total respiration rate (V t) in flower buds showed to be double hump-shaped curves. Short day raised, brought the first-hump of V t forward and delayed the second-hump, while long day delayed the whole curve. The capacity (V alt) and activity (ρV alt) of SD and LD changed synchronously and both showed to be double hump-shaped curves. Short day made the first climax of V alt and pV alt existed much earlier, while long day increased their rates significantly. The length of day had little effects on the later period climax. Long day also increased the contributions of alternative respiration pathway in total respiration rate (ρV alt/V t). The changes in alternative respiration pathway were correlated with the induction of dormancy and adjusted by photoperiod. Short day promoted dormancy induction of nectarine trees, while long day delayed it.

Reversibility of the Resistance to Lenalidomide and Pomalidomide and Absence of Cross-Resistance in a Murine Model of MM

Abstract 134 Introduction:The introduction of novel agents has improved the outcome of MM patients, but MM is still considered an incurable disease and the emergence of resistance is the main responsible for this situation. The unraveling of the resistance mechanisms would help to design novel therapeutic strategies (combinations or sequencing treatments) to overcome this problem. Material and Methods:We have developed an in vivo model of acquired resistance to antimyeloma agents based in a model of subcutaneous plasmocytoma (MM1S) in CB17-SCID mice. For this purpose, mice were treated with Lenalidomide (25 mg/Kg) + Dex (1 mg/Kg) (LD), Pomalidomide (5 mg/Kg) + Dex (PD) or vehicle control, and after a period of initial sensitivity, tumors developed resistance to the administered combination. At this moment selected sensitive and resistant tumors were excised to analyze apoptosis, signaling pathways and gene expression profiling (GEP) changes. Moreover, some of these mice bearing resistant tumors were switched to receive the alternative combination (that is LD-PD or PD- LD), and, in selected mice, a second change of treatment was performed after secondary resistance to receive again the initial treatment (LD-PD-LD or PD-LD-PD). In order to evaluate TTP and to define the moment to change treatment we considered progression when tumor volume reached 1.700 mm3. Results:Both combinations (LD & PD) controlled the initial growth of the tumors, with a higher potency for the PD combination. Tumor volume reached 500 mm3 at a median of 8, 42 & 53 days for control, LD and PD respectively (p=0.01 and p=0.001). Nevertheless, after 30 days of continuous treatment, and despite maintaining the administration of the drugs, tumors started growing, and, once the tumors had reached 500 mm3, their growth kinetic was similar for the treated mice (despite still being treated) as compared to the untreated mice, indicating the emergence of complete resistance. This resistance was also confirmed ex vivo by in vitro culture with the corresponding drugs.In order to test the presence of cross-resistance, mice bearing big tumors resistant to LD or PD and already growing in an exponential phase (volume of 1.700 mm3), were at this point treated with the alternative combination. This sequential treatment change induced tumor stabilization and even a decrease of tumor volume. Again PD was significantly more potent at overcoming LD resistance as compared to the alternative situation, and this was verified both in terms of tumor growth inhibition (p=0.005) and in terms of time to progression (median of 16 vs 27 days for LD and PD respectively. p=0.004).Furthermore, mice that had been treated with LD-PD or PD-LD and had developed resistance to both combinations were again treated with the initial combination, and, surprisingly, they were again sensitive, indicating the reversibility of the acquired resistance. Similarly to previous experiments, PD was again significantly more potent than LD. This reversibility was also confirmed ex vivo after culture of the cells in medium without drugs.In order to investigate the resistance mechanisms, cells extracted from sensitive and resistant tumors were analyzed by Western Blot. Treatment with LD and PD induced a downregulation of pERK 1/2, nevertheless, when these cells developed resistance a very significant increase of pERK 1/2 levels (even higher than the basal levels) was observed. Moreover, these resistant cells also showed an upregulation of p-MEK, p-RAF and RAS. In this same line, the MEK inhibitor PD-98059 potentiated the in vitro activity of LD or PD in fresh MM1S cells, with high synergism (CI<0.1).Finally, changes in GEP were evaluated in extracted tumors. A significant change was observed in cells from tumors sensitive to PD and LD (with a higher gene deregulation with the former combination), as compared to untreated ones. Interestingly, when tumors became resistant, most of these changes disappeared and the GEP partially returned to a profile similar to that of active untreated tumors. Conclusion:The data presented would support treatment with alternative IMIDs if resistance was developed to one of them (specially PD was significantly more potent at rescuing resistance to LD), or even the retreatment with the same IMID after a wash up period. Moreover this study supports the evaluation of combinations of IMIDs with agents that abrogate the ERK pathway in order to increase efficacy or avoid resistance. Disclosures:Quintana:Celgene: Employment. García:Celgene: Employment. Pandiella:Celgene: Research Funding. San Miguel:Celgene: Consultancy, Research Funding.

Obesity alters circadian behavior and metabolism in sex dependent manner in the volcano mouse Neotomodon alstoni

The aim of the present study is to evaluate whether circadian locomotor activity, and the daily profile of plasma parameters related to metabolic syndrome (nutrients: glucose and triacylglycerides, and hormones: insulin and leptin), differ between male and female Neotomodon alstoni mice, both lean and obese. Young adult animals were captured in the field and kept at the laboratory animal facility. After 6 to 7months feeding the animals ad libitum with a regular diet for laboratory rodents, 50–60% of mice became obese. Comparisons between sexes indicated that lean females were more active than males; however obese females reduced their nocturnal activity either in LD or DD, and advanced the phase of their activity-onset with respect to lights off. No differences in food intake between lean and obese mice, either during the day or night, were observed. Daily profiles of metabolic syndrome-related plasma parameters showed differences between sexes, and obesity was associated with increased values, especially leptin (500% in females and 273% in males) and insulin (150% in both females and males), as compared with lean mice. Our results indicate that lean mice display behavioral and endocrine differences between sexes, and obesity affects the parameters tested in a sex-dependent manner. The aforementioned leads us to propose N. alstoni, studied in captivity, could be an interesting model for the study of sex differences in the effects of obesity.

Dynamic Mobility of Immunological Cells Expressing S100A8 and S100A9 in vivo: A Variety of Functional Roles of the two Proteins as Regulators in Acute Inflammatory Reaction

The immunological properties of rat S100A8 (r-S100A8) and S100A9 (r-S100A9) in immune cells are poorly understood. Enzyme-linked immunosorbent assay (ELISA) for r-S100A9 enabled us to discuss the differential functional roles of the two proteins, and their localization in the cells was observed microscopically. Recombinant human S100A8 (rh-S100A8) or S100A9 (rh-S100A9) were intravenously administrated into rats with LPS-induced liver damage. ELISA was used to measure the serum concentration of S100A9 in the rats. Western blotting and a preparative ELISA were used to prove specificity and avidity of monoclonal antibodies for r-S100A8 and r-S100A9. Immunohistochemical staining was carried out to visualize intracellular localization of the two proteins in the immune cells using the antibodies. When rh-S100A8 was intravenously injected in the rats (B group), the serum concentration of r-S100A9 apparently decreased as compared with that of the positive control rats (A group). The activities of AST, ALT, and LD in the rat sera (B group) also significantly went down in comparison with those of the rats (A group). Although both the S100A8 and S100A9 were abundantly expressed in activated immune cells, quite difference of not only their intracellular localization but also distribution of the cells expressing the two proteins was microscopically observed. In the rats (B group), less number of the immune cells or less amount of r-S100A8 and r-S100A9 in the cells than those of the rats (A group) was also seen. The r-S100A8 could serve as a regulator of acute inflammatory reaction in the rats with LPS-induced damage.

Artificial long days and daily contact with bucks induce ovarian but not oestrous activity during the non-breeding season in Mediterranean goat females

The aim of the present study was to determine whether a treatment of 3 mo of artificial long days and daily contact with bucks can stimulate reproductive activity during the normal seasonal anoestrous in female goats, and whether such treatment modifies the onset of the normal breeding season. Thirty-nine adult, open does were assigned to two treatments of similar mean body weight (BW) and body condition score (BCS). One treatment (LD; n = 18) was housed in a light-proof building and exposed to long days (16 h of light/d) from 17 November to 5 February, and then exposed to the natural photoperiod in an open shed. The remaining females were housed in an open shed under natural photoperiod conditions throughout the experiment (control [C]; n = 21). Plasma samples for progesterone, BW and BCS were recorded every wk. Oestrous activity was checked daily using aproned bucks. Bucks were housed close to females in a separate barn from the onset of the experiment. Ovulation rate was determined by laparoscopy 7 d after positive identification of oestrus. The interaction of treatment by time for temporal concentrations patterns of progesterone concentrations indicated that luteal activity in LD does were greater ( P < 0.001) than those of C does during the natural seasonal anoestrous season. None of the C does exhibited oestrous or luteal activity during the non-breeding season; whereas, 72% of LD does exhibited luteal activity only 33% of them showed oestrous activity and during this season ( P < 0.01). Differences in resumption of the oestrous or luteal activity were not observed ( P > 0.05) in the subsequent breeding season between treatments. In conclusion, 3 mo of exposing does to long days and daily contact with bucks during the breeding season appears to stimulate reproductive processes that normal would not occur during the anoestrous season. However, this treatment does not induce oestrus is adequate numbers of does to be of practical value. Finally, this treatment does not modify the onset of the subsequent natural breeding season.

Immunocytochemical evidence for different patterns in daily rhythms of VIP and AVP peptides in the suprachiasmatic nucleus of diurnal Funambulus palmarum

The suprachiasmatic nucleus (SCN) is the principal pacemaker that coordinates circadian rhythmicity in mammals. The studies on understanding the circadian system in diurnal rodents are limited. In this study, we have used the 3 striped South Indian Palm Squirrel (Funambulus palmarum). The locomotor activity showed a diurnal pattern of activity in LD 12:12, constant darkness (DD) and light (LL) conditions with circadian periods (τ) of 24.19±0.1, 24.11±0.03 and 24.92±0.35h respectively. Anatomical study of the brain revealed that this animal had short, thick and stout optic nerves with SCN elliptical in shape with a higher neuronal population as distinct from nocturnal rodents. Since the neuropeptides, vasoactive intestinal polypeptide (VIP) and arginine vasopressin (AVP) play important roles in photic entrainment and relay of information respectively in nocturnal rodents, we studied the distribution and daily rhythms of VIP-ir and AVP-ir in squirrel SCN. The VIP-ir and AVP-ir cells in the SCN showed a ventrolateral and dorsomedial distribution with daily rhythmicity in their levels. The peak time of VIP-ir rhythm was found ahead of AVP-ir. The VIP-ir levels were higher for longer duration than AVP-ir levels. The maximum and minimum VIP-ir levels were at ZT-6 and ZT-0 respectively and AVP-ir levels at ZT-12 and ZT-0 respectively. Thus, VIP and AVP maximum and minimum levels appeared 6 and 12h apart respectively in squirrel, though 12 and 8h apart in rat. These findings in the present report could be a step towards underpinning the mechanisms regulating diurnality.

Processing Circadian Data Collected from the Drosophila Activity Monitoring (DAM) System: Figure 1.

Adult behavioral assays have been used with great success in Drosophila melanogaster to identify circadian rhythm genes. In particular, the locomotor activity assay can identify altered behavior patterns over the course of several days in small populations, or even individual flies. Generally, circadian behavior is assayed during a period of 12 h light:12 h dark cycling (LD entrainment) followed by conditions of constant darkness (DD). LD activity profiles provide a qualitative image of daily activity bouts, and the data can be used to quantitatively assess the phase and/or amplitude of particular bouts. Additional activity assessments made from entrained flies that have been shifted to constant darkness can provide insight into the state of internal clocks and the ability of these clocks to drive rhythmic outputs. Typical LD DD runs assess both free-running rhythmicity and period length with χ2 periodogram (P'gram) analysis. This protocol describes the use of ClockLab (a MATLAB-based program) and the Counting Macro (an Excel-based program) to analyze circadian locomotor activity data collected using the Drosophila Activity Monitoring (DAM) System from TriKinetics. Specific procedures are described to analyze free-running rhythmicity and period length for individual flies, and assess group activity plots during both entrainment and constant conditions.

Preclinical toxicological assessment of a phytotherapeutic product – CPV (based on dry extracts of Crataegus oxyacantha L., Passiflora incarnata L., and Valeriana officinalis L.)

Associations of plants have been widely used, for centuries, in Ayurveda and in Chinese medicine and have been increasingly acknowledged in Western medicine. The objective of this study is to assess the level of toxicity of an association of three plants: Crataegus oxyacantha, Passiflora incarnata, and Valeriana officinalis (CPV extract). This association was administered to rats, mice, and dogs, both acute and chronically for 180 days. The tests used in the acute experiments were: observational pharmacological screening, LD(50), motor coordination and motor activity. Chronic tests carried out were: weight gain/loss and behavioral parameters in rats and in mice; estrus cycle, effects on fertility, and teratogenic studies in rats and of mutagenic features in mice, in addition to the Ames test. The following parameters were assessed in dogs: weight gain/loss, general physical conditions, water/food consumption and anatomopathological examination of the organs subsequent to the 180 days of treatment. All of the results were negative, showing that CPV administered in high doses and over a long period of time presents no toxicity, suggestive of the fact that this is an association devoid of risk for human beings.

A Case of IgG kappa-LD Complex, Producing Long-Term Increase of Enzyme Activity and a Rare Isoenzyme Pattern

Background: Serum LD activity is increased in several diseases. The clinical significance of elevated LD may be evaluated by isoenzyme analysis. Here, we report on a long-term elevation of serum LD activity with a rare isoenzyme pattern caused by LD-IgG complexes. Patient, methods: A 75-year old female with atherosclerosis and implantations of two stents showed increased LD activity and an unusual isoenzyme pattern. LD activity, inhibition and immunoglobulins were measured with Roche tests; immunofixation was performed with a Helena test. LD isoenzymes were separated in agarose gel and quantified by the color formed by NADH, reduced phenazine methosulfate and nitro blue tetrazolium. Results: LD isoenzyme analyses revealed one band near the slowly migrating LD-4 position. The band contained LD complexed with immunoglobulins (IgG with κ light chains), The long-term increased serum LD enzyme activity in this patient may have been due to abnormally slow clearance of these large macro-LD complexes from circula...

The barley limit dextrinase inhibitor: Gene expression, protein location and interaction with 14-3-3 protein

The barley ( Hordeum vulgare L.) limit dextrinase inhibitor (LDI) binds to and prevents the enzymic action of the starch debranching enzyme limit dextrinase (E.C. 3.2.1.142), and reduced levels of LDI have pronounced effects on starch biosynthesis including a loss of the small B-type starch granules. In this work, the time and place of LDI gene expression, and also the expression of LDI protein and inhibitory activity, and binding of LDI to the regulatory protein 14-3-3 were investigated. It was found that LDI is encoded for by a small multigene family, and the LDI gene is expressed at 2–4 weeks post anthesis in the endosperm. The LDI protein is present and active from 4 weeks post anthesis onward only in the starchy endosperm, not in the aleurone layer or other tissues, and is broken down during germination. Immunolocalisation shows the LDI protein to accumulate predominantly in the outer regions of the starchy endosperm, which coincides with the region where the small B-type granules are found. Additionally, the LDI protein sequence contains a 14-3-3 binding motif, and LDI binds to the barley 14-3-3A and 14-3-3C proteins in a phosphorylation-dependent manner, although addition of full length 14-3-3 proteins had no effect on LDI or LD activities in vitro. These findings are consistent with an important role for LDI (and by inference limit dextrinase) in the regulation of starch biosynthesis and starch granule formation.