Evidence of a role for cyclic ADP-ribose in calcium signalling and neurotransmitter release in cultured astrocytes

Astrocytes possess different, efficient ways to generate complex changes in intracellular calcium concentrations, which allow them to communicate with each other and to interact with adjacent neuronal cells. Here we show that cultured hippocampal astrocytes coexpress the ectoenzyme CD38, directly involved in the metabolism of the calcium mobilizer cyclic ADP-ribose, and the NAD+ transporter connexin 43. We also demonstrate that hippocampal astrocytes can release NAD+ and respond to extracellular NAD+ or cyclic ADP-ribose with intracellular calcium increases, suggesting the existence of an autocrine cyclic ADP-ribose-mediated signalling. Cyclic ADP-ribose-induced calcium changes are in turn responsible for an increased glutamate and GABA release, this effect being completely inhibited by the cyclic ADP-ribose specific antagonist 8-NH2-cADPR. Furthermore, addition of NAD+ to astrocyte-neuron co-cultures results in a delayed intracellular calcium transient in neuronal cells, which is strongly but not completely inhibited by glutamate receptor blockers. These data indicate that an astrocyte-to-neuron calcium signalling can be triggered by the CD38/cADPR system, which, through the activation of intracellular calcium responses in astrocytes, is in turn responsible for the increased release of neuromodulators from glial cells.     

Role of a novel maintained low-voltage-activated inward current permeable to sodium and calcium in pacemaking of insect neurosecretory neurons

Among ionic currents underlying neuronal pacemaker activity, low-threshold-activated calcium currents contribute to setting the threshold for spike firing. In the insect central nervous system, dorsal unpaired median (DUM) neurons are capable of generating spontaneous electrical activity. It has previously been shown that two distinct (transient and maintained) low-voltage-activated (LVA) calcium currents are responsible for the generation of the pacemaker potential. Whole-cell recordings in voltage- and current-clamp mode were obtained from short-term cultured DUM neurons. Using 100 mM sodium and 2 mM calcium as charge carrier in the external solution as well as conditions that eliminate calcium currents (0.5 mM CdCl₂), voltage-clamp experiments showed that a hitherto unanticipated LVA maintained inward current, activated at around −60 mV, was present. The current amplitude was strongly dependent on internal ATP concentration. Sodium-free solution reduced by 80% the current amplitude. Increasing (5 mM) or decreasing (calcium-free) external calcium concentrations enhanced or reduced, respectively, the maximum conductance without any effect on the voltage dependence. This novel ion channel was permeable to barium but manipulating internal or external magnesium concentrations was without effect on current amplitude or reversal potential. Based on IC₅₀ values, the maintained current was 50-fold less sensitive to TTX than the classical transient voltage-dependent sodium current. Furthermore, it was insensitive to ethosuximide and halothane. Voltage-dependent inactivation analysis revealed an unexpected calcium-sensitive process that involved calcineurin. From these results it appears that, besides the two LVA calcium currents previously described, another LVA maintained inward current permeable to both sodium and calcium was also involved in the generation of the predepolarization. Based on these findings, we propose that a novel calcium-dependent mechanism is involved in the regulation of the pacemaker activity.     

The effects of disodium pamidronate on human polymorphonuclear leukocytes and platelets: An <Emphasis Type="Italic">in vitro</Emphasis> study

Recent reports have indicated that, as well as having antiresorptive effects, bisphosphonates could have an application as anti-inflammatory drugs. Our aim was to investigate whether this anti-inflammatory action could be mediated by the nitric oxide (NO) released by the leukocytes migrating to the site of inflammation. In particular, we investigated in vitro the intracellular calcium concentration ([Ca²⁺]_i), the level of NO released by PMN and platelets, and the PMN myeloperoxidase activity after incubation with disodium pamidronate, since there was a postulated modulatory effect of this aminosubstituted bisphosphonate on leukocytes both in vitro and in vivo. Our data shows that the pamidronate treatment provoked a significant increase in the [Ca²⁺]_i parallel to the enhancement in NO release, suggesting a possible activation of constitutive nitric oxide synthase, while the myeloperoxidase activity was significantly reduced. In conclusion, we hypothesized that treatment with pamidronate could stimulate NO-production by cells present near the bone compartment, thus constituting a protective mechanism against bone resorption occurring during inflammation. In addition, PMN- and platelet-derived NO could act as a negative feed-back signal to restrict the inflammatory processes.     

Changes in the level of cytosolic calcium, nitric oxide and nitric oxide synthase activity during platelet aggregation: an in vitro study in platelets from normal subjects and those with cirrhosis

Variceal bleeding due to abnormal platelet function is a well-known complication of cirrhosis. Nitric oxide-related stress has been implicated in the pathogenesis of liver cirrhosis.In the present investigation,we evaluated the level of platelet aggregation and concomitant changes in the level of platelet cytosolic calcium (Ca2+), nitric oxide (NO) and NO synthase (NOS) activity in liver cirrhosis.The aim of the present study was to investigate whether the production of NO by NOS and level of cytosolic Ca2+ influence the aggregation of platelets in patients with cirrhosis of the liver.Agonist-induced aggregation and the simultaneous changes in the level of cytosolic Ca2+, NO and NOS were monitored in platelets of patients with cirrhosis.Platelet aggregation was also measured in the presence of the eNOS inhibitor,diphenylene iodinium chloride (DIC).The level of agonist-induced platelet aggregation was significantly low in the platelets of patients with cirrhosis compared with that in platelets from normal subjects.During the course of platelet aggregation,concomitant elevation in the level of cytosolic Ca2+ was observed in normal samples,whereas the elevation was not significant in platelets of patients with cirrhosis.A parallel increase was observed in the levels of NO and NOS activity.In the presence of the eNOS inhibitor,platelet aggregation was enhanced and accompanied by an elevated calcium level.The inhibition of platelet aggregation in liver cirrhosis might be partly due to greater NO formation by eNOS.Defective Ca2+ release from the internal stores to the cytosol may account for inhibition of aggregation of platelets in cirrhosis.The NO-related defective aggregation of platelets in patients with cirrhosis found in our study is of clinical importance,and the underlying mechanism of such changes suggests a possible therapeutic strategy with cell-specific NO blockers.     

A possible calcium binding site in D1 protein: A fluorescence and FTIR study of the interaction between lanthanides and a synthetic peptide

A peptide ranging from residue 229 to 240 of the D₁ protein of Photosystem (PS) II was synthesized and lanthanides were used as candidates of calcium. Fluorescence and FTIR spectroscopy were used to test the conformational adaptation after lanthanide additions. Fluorescence spectroscopy showed that the synthetic peptide provides lanthanide binding site, and that glutamic acids are involved in lanthanide binding. Resolution enhancement techniques were combined with band curve-fitting procedures to quantitate the FTIR spectral information from the amide 1 bands. The relative areas of these component bands indicate that lanthanide induced a substantial decrease in the amount of unordered structure and turns, while a corresponding increase in the amount of -helix and open loop was also observed. This indicates that a relatively compact structure of the synthetic peptide is formed if lanthanides are applied. The results may reflect on the physiological and biochemical function of calcium in PS II, including preventing D₁ from trypsin digestion.Abbreviations DCMU 3-(3,4-Dichlorophenyl)-1,1-dimethylurea - FTIR Fourier transform infrared - FSD Fourier self-deconvolution - PS Photosystem - Q_B Secondary plastoquinone electron acceptor of PS II     

Internal desynchronization of the circadian activity and feeding rhythm in an owl monkey (Aotus lemurinus griseimembra): a case study

In the free-running circadian locomotor activity rhythm of a 7-year-old male owl monkey (Aotus lemurinus griseimembra) kept under constant light and climatic conditions (LL 0.2 lux, 25°C ± 1°C, 60 ± 5% relative humidity [RH]), a second rhythm component developed that showed strong relative coordination with the free-running activity rhythm of 24.4h and a 24h rhythm. The simultaneously recorded feeding activity rhythm strongly resembled this rhythm component. Therefore, it seems justified to infer that there was an internal desynchronization between the two behavioral rhythms or their circadian pacemakers, that is, between the light-entrainable oscillator located in the suprachiasmatic nuclei (SCN) and a food-entrainable oscillator located outside the SCN. This internal desynchronization may have been induced and/or maintained by a zeitgeber effect of the (irregular) 24h feeding schedule on the food-entrainable oscillator. The weak relative coordination shown by the activity rhythm indicates a much weaker coupling of the light-entrainable oscillator to the food-entrainable oscillator than vice versa. (Chronobiology International, 17(2), 147-153, 2000)     

Hospital cardiovascular deaths and total distribution of deaths in 180 consecutive months with different cosmic physical activity: a correlative study (1974–1988)

The dynamics of total hospital deaths from different kinds of cardiovascular diseases in one 1000-bed hospital were compared with 10 monthly cosmic/solar and geomagnetic physical activity parameters. Data used were of 180 consecutive months; 15601 deaths including 5667 from cardiovascular diseases were included in this study. It was concluded that the number of monthly hospital deaths shows a highly significant correlation with monthly solar physical activity.     

The difference between in bed and out of bed activity as a behavioral marker of cancer patients: A comparative actigraphic study

The current study was conducted to provide normative data on actigraphic dichotomy index (I?<?O) (the percentage of in bed activity counts that are less than the median of out of bed counts) in healthy population and to assess whether the I?<?O could be an effective index in discriminating the circadian motor activity of cancer patients from healthy controls. In this retrospective study, we recovered 408 actigraphic records from two databases: healthy controls (n?=?182; 79 males; mean age 38.7?±?12.6) and patients with metastatic colorectal cancer (n?=?226; 149 males; mean age 58.4?±?11.4). Beside the usual actigraphic sleep parameters (time in bed, sleep onset latency, total sleep time, wake after sleep onset, sleep efficiency, number of awakenings, and mean motor activity), we also computed the dichotomy index and number of actigraphic wake parameters, namely, diurnal motor activity, diurnal total sleep time, number of sleep episodes, and the mean duration of the longest diurnal sleep episode. Using the Youden index, we calculated the cut off value that performed the best for I?<?O and actigraphic wake parameters. Finally, we created Receiver Operator Characteristic curves to test the efficacy of each actigraphic parameter to discriminate cancer patient from healthy controls. Mean I?<?O was 99.5% (SD, 0.48%) in the healthy group, as compared to 96.6% (SD, 3.6%) in the cancer group (p?<?0.0001). Important age-related effects appeared unlikely after performing both the main analysis with age as a covariate, and a subset analysis in 104 subjects matched for age and sex. In the main analysis, all actigraphic parameters, except total sleep time, significantly differentiated the two groups of participants. However, the I?<?O was the one that clearly performed best. Here, we provide the first large dataset on I?<?O in healthy subjects, we confirm the relevance of this circadian index for discriminating advanced stage colorectal cancer patients from healthy subjects, and we lay the grounds for further investigations of this circadian index in patients with other chronic diseases.     

Decline of tactile acuity in aging: a study of body site,blood flow,and lifetime habits of smoking and physical activity

Tactile acuity of 60 older subjects (≥?65 years) and 19 younger subjects (18–28 years) was assessed by two-point gap thresholds at the upper and lower surfaces of the forefinger, at the upper and lower surfaces of the feet, and at the volar surface of the forearm. The older subjects were assigned to one of four groups of 15 subjects each, depending on reported lifetime habits of physical activity and smoking: (1) active smokers, (2) active nonsmokers, (3) inactive smokers, and (4) inactive nonsmokers. Peripheral blood flow was assessed at the forefinger, foot, and forearm by means of laser-Doppler imaging and skin temperature recordings, under resting conditions and during and after a 5-min exposure to mild cooling (28°C). Consistent with previous studies, tactile acuity thresholds in the foot and finger averaged about 80% higher in the older subjects than in the younger subjects, but only about 22% higher in the forearm. Although the upper surface of the fingertip was more sensitive than the lower surface in both younger and older subjects, the age-related decline in tactile acuity was nearly identical on both sides of the finger and foot. The latter finding refutes the hypothesis that the larger effect of aging in the extremities results from greater physical wear and tear on the contact surfaces of the hands and feet. Self-reported lifetime histories of physical activity and smoking were not significantly associated with measures of cutaneous blood flow or tactile thresholds. Possible reasons for this lack of association are discussed, including the inherent limitations of testing only healthy older subjects, and the concept of “successful aging”.     

Gastrin-releasing peptide receptor gene silencing inhibits the development of the epithelial–mesenchymal transition and formation of a calcium oxalate crystal in renal tubular epithelial cells in mice with kidney stones via the PI3K/Akt signaling pathway

Between 1% and 15% of people are globally affected by kidney stones, and this disease has become more common since the 1970s. Therefore, this study aims to investigate the effects of gastrin-releasing peptide receptor (GRPR) gene silencing via the PI3K/Akt signaling pathway on the development of the epithelial–mesenchymal transition (EMT) and formation of a calcium oxalate crystal in renal tubular epithelial cells (TECs) of kidney stones. A total of 70 clean and healthy C57BL/6J mice were assigned into the normal ( n = 10) and kidney stones groups ( n = 60). The underlying regulatory mechanisms of GRPR were analyzed in concert with the treatment of shGRPR-1, LY294002, and shGRPR-1 + LY294002 in TECs isolated from mice with kidney stones. A series of experiments were conducted for the measurement of urinary oxalate and urinary calcium, the renal calcium salt deposition, the positive rate of GRPR, the expressions of renal TECs related genes and calcium oxalate regulation related genes, and the growth of calcium crystals induced by cells. After treatment of shGRPR-1 and shGRPR-1 + LY294002, levels of urinary oxalate and urinary calcium in the serum, as well as positive rate of GRPR, became relatively low, levels of E-cadherin enhanced, whereas levels of Akt, PI3K, GRPR, extents of PI3K and Akt phosphorylation, α-SMA, Vimentin and FSP-1, OPN, MCP-1, and CD44 decreased and a number of crystals reduced. Taken together, we conclude that GRPR gene silencing suppresses the development of the EMT and formation of the calcium oxalate crystal in renal TECs of kidney stones through the inactivation of the PI3K/Akt signaling pathway.     

传统文化和社会经济活动对生物保护的正面影响: 来自西藏黑白仰鼻猴的实例研究(英文)

黑白仰鼻猴(Rhinopithecus bieti)是仅存于滇西北和藏东南的横断山脉的濒危灵长类动物,目前仅有野外15群约2500只个体,并且西藏红拉雪山自然保护区的小昌都群也许是自1988年调查以来唯一数量保持稳定的种群。通过调查访问、以及分析社会经济数据中隐含的保护信息,该文通过实例研究探讨了传统文化和社会经济发展模式对红拉雪山自然保护区小昌都猴群的影响,试图探讨传统文化和人类活动对该猴群种群保持稳定的正面影响:1)研究结果表明传统的生产方式对黑白仰鼻猴的生境保护有益,例如主要依靠畜牧业和非木材的林产品而不是毁林开荒种植青稞;2)受印度教和佛教影响的藏传佛教对生灵的敬畏,避免当地村民对黑白仰鼻猴的猎杀;3)一妻多夫的大家庭,其人均资源消耗(特别是薪柴,建房木材等)明显比一夫一妻的小家庭少,这种婚俗也许可以缓解人类索取相应自然资源时对黑白仰鼻猴的生境破坏。因此,考虑到小昌都是唯一种群稳定的群,我们认为是当地的社会生产方式、宗教文化以及婚俗习惯让小昌都群长期维持在该生境的最大容纳量。     

Immune function of patients receiving recombinant human interleukin-6 (IL-6) in a phase I clinical study: Induction of C-reactive protein and IgE and inhibition of natural killer and lymphokine-activated killer cell activity

Interleukin-6 (IL-6) is a cytokine that acts on a variety of cell types, including myeloid progenitor cells and B and T lymphocytes. It has been found to activate cytotoxic T cells and natural killer (NK) cells and to induce T-cell-mediated antitumour effects in animal models. In a phase I clinical trial of recombinant human IL-6, 20 patients with advanced cancer were entered to receive daily subcutaneous injections of IL-6 over 7 days followed by a 2-week observation period and another 4 weeks of daily IL-6 injections. Doses varied between 0.5 g/kg and 20 g/ kg body weight and immune functions were monitored throughout. At all dose levels IL-6 administration led to a marked increase in serum levels of C-reactive protein and a moderate rise in complement factor C3. The proportions of CD4, CD8 or HLA-DR lymphocytes in peripheral blood did not alter with IL-6 treatment nor did the in vitro proliferation of peripheral blood mononuclear cells induced by either phytohaemagglutinin, pokeweed mitogen or fixedStaphylococcus aureus. By contrast, NK cell activity, lymphokine-activated killer (LAK) cell activity and proliferation induced by in vitro culture with interleukin-2 (IL-2) were suppressed at doses exceeding 2.5 g/kg. Serum IgE levels were consistently elevated over the IL-6 dose range but IgM, IgG and IgA levels were unaffected. In summary there is a dose-dependent induction of acutephase proteins by in vivo IL-6 treatment. At higher IL-6 doses there is a suppressive effect on NK and LAK activity measured in vitro. IL-6 may thus be useful in combination cytokine therapies that seek to suppress LAK and favour cytotoxic T lymphocyte responses. The rise in IgE levels in response to IL-6 was unexpected and suggests a more pivotal role than previously known for the control of IgE production; this could include IgE-related diseases.Supported by a Mildred Scheel Research-Fellowship from the Deutsche Krebshilfe     

A study of the effect on nucleophilic hydrolytic activity of pancreatic elastase, trypsin, chymotrypsin, and leucine aminopeptidase by boronic acids in the presence of arabinogalactan: a subsequent study on the hydrolytic activity of chymotrypsin by boronic acids in the presence of mono-, di-, and trisaccharides

The hydrolytic activity of trypsin, chymotrypsin, elastase, and leucine aminopeptidase, is inhibited by different boronic acids. However, all the enzymes are inhibited by the compound CbzAla(boro)Gly(OH)(2). Therefore, these additives can control the nucleophilic hydrolytic activity of these enzymes.     

In search of better strategies of using activity traps to collect Dytiscidae and Noteridae (Coleoptera): a case study of a local fauna in central European Russia

Activity traps (ATs) are a widely used method of sampling water beetles (Coleoptera). This case study attempts to investigate the effect of mouth size and trapping effort on the performance of the ATs and reveal sex ratios and proportions of morphs in dimorphic females in populations of Dytiscidae and Noteridae in one particular area of Udomelsky District, Tver Oblast, Russia. ATs are more selective compared to hand netting but make it possible to collect a greater number of abundant species with relatively little expenditures of effort and time. The width of the trap mouth has no effect on efficiency. Long-time (48?h exposure) traps are not more efficient than short-time (24?h) ones, in terms of the relative intensity of collecting individuals and species. The applied number of ATs (up to 330 trap-days) was not sufficient to reveal the full diversity of diving beetles in the studied waterbodies.     

DNA methylation‐based biomarkers of aging were slowed down in a two‐year diet and physical activity intervention trial: the DAMA study

Several biomarkers of healthy aging have been proposed in recent years, including the epigenetic clocks, based on DNA methylation (DNAm) measures, which are getting increasingly accurate in predicting the individual biological age. The recently developed “next‐generation clock” DNAmGrimAge outperforms “first‐generation clocks” in predicting longevity and the onset of many age‐related pathological conditions and diseases. Additionally, the total number of stochastic epigenetic mutations (SEMs), also known as the epigenetic mutation load (EML), has been proposed as a complementary DNAm‐based biomarker of healthy aging. A fundamental biological property of epigenetic, and in particular DNAm modifications, is the potential reversibility of the effect, raising questions about the possible slowdown of epigenetic aging by modifying one''s lifestyle. Here, we investigated whether improved dietary habits and increased physical activity have favorable effects on aging biomarkers in healthy postmenopausal women. The study sample consists of 219 women from the “Diet, Physical Activity, and Mammography” (DAMA) study: a 24‐month randomized factorial intervention trial with DNAm measured twice, at baseline and the end of the trial. Women who participated in the dietary intervention had a significant slowing of the DNAmGrimAge clock, whereas increasing physical activity led to a significant reduction of SEMs in crucial cancer‐related pathways. Our study provides strong evidence of a causal association between lifestyle modification and slowing down of DNAm aging biomarkers. This randomized trial elucidates the causal relationship between lifestyle and healthy aging‐related epigenetic mechanisms.     

Development and validation of a gas chromatography/ion trap-mass spectrometry method for simultaneous quantification of cocaine and its metabolites benzoylecgonine and norcocaine: application to the study of cocaine metabolism in human primary cultured renal cells

Acute renal failure is a common finding in cocaine abusers. While cocaine metabolism may contribute to its nephrotoxic mechanisms, its pharmacokinetics in kidney cells is hitherto to be clarified. Primary cultures of human proximal tubular cells (HPTCs) provide a well-characterized in vitro model, phenotypically representative of HPTCs in vivo. Thus, the present work describes the first sensitive gas chromatography/ion trap-mass spectrometry (GC/IT-MS) method for measurement of cocaine and its metabolites benzoylecgonine (BE) and norcocaine (NCOC) using a primary culture of HPTCs as cellular matrix, following solid phase extraction (SPE) and derivatization with N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA). The application of this methodology also enables the identification of two other cocaine metabolites: ecgonine methyl ester (EME) and anhydroecgonine methyl ester (AEME). The validation of the method was performed through the evaluation of selectivity, linearity, precision and accuracy, limit of detection (LOD), and limit of quantification (LOQ). Its applicability was demonstrated through the quantification of cocaine, BE and NCOC in primary cultured HPTCs after incubation, at physiological conditions, with 1 mM cocaine for 72 h. The developed GC/IT-MS method was found to be linear (r2 > 0.99). The intra-day precision varied between 3.6% and 13.5% and the values of accuracy between 92.7% and 111.9%. The LOD values for cocaine, BE and NCOC were 0.97±0.09, 0.40±0.04 and 20.89±1.81 ng/mL, respectively, and 3.24±0.30, 1.34±0.14 and 69.62±6.05 ng/mL as LOQ values.