Thrombin-induced phosphorylation of MARCKS does not alter its interactions with calmodulin or actin

Myristoylated alanine-rich C kinase substrate (MARCKS) is a calmodulin (CaM)- and actin-binding protein and prominent protein kinase C (PKC) substrate. In vitro phosphorylation of MARCKS by PKC has been shown to induce the release of both CaM and actin, leading to the suggestion that MARCKS may regulate CaM availability during agonist-induced signalling. In support of this hypothesis we previously demonstrated that thrombin-induced MARCKS phosphorylation in endothelial cells (EC) parallels activation of myosin light chain kinase, a CaM-dependent enzyme. To test this theory further, we transfected CHO cells, which normally do not express significant levels of MARCKS, with a MARCKS cDNA. The thrombin-stimulated phosphorylation of myosin light chains and the sensitivity to CaM antagonists in the MARCKS overexpressing cells was the same as that in control CHO cells. MARCKS associated with the actin cytoskeleton in EC was markedly increased upon treatment with the PKC activator, PMA, but only modestly enhanced by thrombin treatment. Similarly, colocalisation of MARCKS with actin was enhanced when the EC were challenged with PMA but not thrombin. These data may be partially explained by PKC-independent phosphorylation of MARCKS in response to thrombin stimulation.     

G protein beta2 subunit interacts directly with neuropathy target esterase and regulates its activity

Neuropathy target esterase (NTE) was identified as the primary target of organophosphate compounds that cause a delayed neuropathy with degeneration of nerve axons. NTE is a novel phospholipase B anchored to the cytoplasmic face of endoplasmic reticulum and essential for embryonic and nervous development. However, little is known about the regulation of NTE. A human fetal brain cDNA library was screened for proteins that interact with NTE, Gbeta2 and Gbeta2-like I subunits were found to be able to bind the C-terminal of NTE in yeast. The interaction of Gbeta2 and NTE was confirmed by in vivo co-immunoprecipitation analysis in COS7 cells. Furthermore, depletion of Gbeta2 by RNA interference down regulated the activity of NTE but not its expression level. In addition, the activity of NTE was down regulated by the G protein signal pathway influencing factor, pertussis toxin, treatment in vivo. These findings suggest that Gbeta2 may play a significant role in maintaining the activity of NTE.     

Pregnenolone protects the PC-12 cell line against amyloid beta peptide toxicity but its sulfate ester does not

Pregnenolone (P), the main precursor of the steroids, and its sulfate ester, pregnenolone sulfate (PS), are the major neurosteroids produced in the neural tissue. Many neuroendocrinological studies stressed the neuroprotective role of neurosteroids although it has been suggested that the inhibition of P and PS synthesis can delay neuronal cell death. The potential roles of P and PS in vital neuronal functions and in amyloid beta peptide (Aβ) toxicity are not clearly identified. This work aims to investigate the effects of P and PS on cell viability and Aβ peptide toxicity in a concentration and exposure time-dependent manner in rat PC-12 cells. The cells were treated with 20 μM Aβ peptide 25-35 and variable concentrations of P and PS ranging from 0.5 μM to 100 μM. To examine the effects of steroid treatment on Aβ peptide toxicity, 0.5 μM (low) and 50 μM (high) neurosteroids were used. The cell viability and lactate dehydrogenase release of cells were evaluated after 24, 48 and 72 h. Morphological changes of cells were also examined. The treatment with higher than 1 μM concentrations of P and PS significantly decreased the cell viability comparing to untreated cells. At lower concentrations, P and PS had no toxic actions until 72 h. The Aβ treatment resulted in a significant decrease in cell viability comparing to untreated cells. P showed a dose-dependent protective effect against Aβ peptide in PC-12 cells. But its sulfate ester did not have the same effect on Aβ peptide toxicity, even it significantly decreased cell viability in Aβ-treated cells. Consequently, the discrepant effects of P and PS on Aβ peptide toxicity may provide insight on the pathogenesis of Alzheimer’s disease.     

Reproductive experience does not persistently alter prefrontal cortical-dependent learning but does alter strategy use dependent on estrous phase

Reproductive experiences in females comprise substantial hormonal and experiential changes and can exert long lasting changes in cognitive function, stress physiology, and brain plasticity. The goal of this research was to determine whether prior reproductive experience could alter a prefrontal–cortical dependent form of learning (strategy set shifting) in an operant box. In this study, female Sprague–Dawley rats were mated and mothered once or twice to produce either primiparous or biparous dams, respectively. Age-matched nulliparous controls (reproductively-naïve females with no exposure to pup cues) were also used. Maternal behaviors were also assessed to determine whether these factors would predict cognitive flexibility. For strategy set shifting, rats were trained in a visual-cue discrimination task on the first day and on the following day, were required to switch to a response strategy to obtain a reward. We also investigated a simpler form of behavioral flexibility (reversal learning) in which rats were trained to press a lever on one side of the box the first day, and on the following day, were required to press the opposite lever to obtain a reward. Estrous phase was determined daily after testing. Neither parity nor estrous phase altered total errors or trials to reach criterion in either the set-shifting or reversal-learning tasks, suggesting that PFC-dependent cognitive performance remains largely stable after 1 or 2 reproductive experiences. However, parity and estrous phase interacted to alter the frequency of particular error types, with biparous rats in estrus committing more perseverative but fewer regressive errors during the set-shifting task. This suggests that parity and estrous phase interfere with the ability to disengage from a previously used, but no longer relevant strategy. These data also suggest that parity alters the behavioral sensitivity to ovarian hormones without changing overall performance.     

Caveolin-1 interacts directly with dynamin-2

Caveolin is the principal component of caveolae in vivo. In addition to a structural role, it is believed to play a scaffolding function to organize and inactivate signaling molecules that are concentrated on the cytoplasmic surface of caveolar membranes. The large GTPase dynamin has been shown to mediate the scission of caveolae from the plasma membrane, although it is unclear if dynamin interacts directly with caveolin or via accessory proteins. Therefore, the goal of this study was to test whether dynamin associates with caveolae via a direct binding to the caveolin 1 (Cav1) protein. Immunoelectron microscopy of lung endothelium or a cultured hepatocyte cell line stained with antibodies for Dyn2 and Cav1 shows that these proteins co-localize to caveolae. To further define this interaction biochemically, in vitro experiments were performed using glutathione-S-transferase (GST)-Dyn2 and GST-Cav1 fusion proteins, which demonstrated a direct interaction between these proteins. This interaction appears to be mediated by the proline-arginine-rich domain (PRD) of Dyn2, as a GST-PRD fragment binds Cav1 while GST-Dyn2DeltaPRD does not. Further, in vitro binding studies using two Dyn2 spliced forms and Cav1 peptides immobilized on paper identify specific domains of Cav1 that bind Dyn2. Interestingly, these Cav1-binding domains differ markedly between two spliced variant forms of Dyn2. In support of these distinctive physical interactions, we find that the different Dyn2 forms, when expressed as GTPase-defective mutants, exert markedly different inhibitory effects on caveolae internalization, as assayed by cholera toxin uptake. These studies provide the first evidence for a direct interaction between dynamin and the caveolin coat, and demonstrate a selectivity of one Dyn2 form toward the caveolae-mediated endocytosis.     

Trigger factor interacts with the signal peptide of nascent Tat substrates but does not play a critical role in Tat-mediated export.

Twin-arginine translocation (Tat)-mediated protein transport across the bacterial cytoplasmic membrane occurs only after synthesis and folding of the substrate protein that contains a signal peptide with a characteristic twin-arginine motif. This implies that premature contact between the Tat signal peptide and the Tat translocon in the membrane must be prevented. We used site-specific photo-crosslinking to demonstrate that the signal peptide of nascent Tat proteins is in close proximity to the chaperone and peptidyl-prolyl isomerase trigger factor (TF). The contact with TF was strictly dependent on the context of the translating ribosome, started early in biogenesis when the nascent chain left the ribosome near L23, and persisted until the chain reached its full length. Despite this exclusive and prolonged contact, depletion or overexpression of TF had little effect on the kinetics and efficiency of the Tat export process.     

Polo-like kinase 4 controls centriole duplication but does not directly regulate cytokinesis

Centrioles organize the centrosome, and accurate control of their number is critical for the maintenance of genomic integrity. Centrioles duplicate once per cell cycle, and duplication is coordinated by Polo-like kinase 4 (Plk4). We previously demonstrated that Plk4 accumulation is autoregulated by its own kinase activity. However, loss of heterozygosity of Plk4 in mouse embryonic fibroblasts has been proposed to cause cytokinesis failure as a primary event, leading to centrosome amplification and gross chromosomal abnormalities. Using targeted gene disruption, we show that human epithelial cells with one inactivated Plk4 allele undergo neither cytokinesis failure nor increase in centrosome amplification. Plk4 is shown to localize exclusively at the centrosome, with none in the spindle midbody. Substantial depletion of Plk4 by small interfering RNA leads to loss of centrioles and subsequent spindle defects that lead to a modest increase in the rate of cytokinesis failure. Therefore, Plk4 is a centriole-localized kinase that does not directly regulate cytokinesis.     

Calmodulin overexpression does not alter Cav1.2 function or oligomerization state

Interactions between calmodulin (CaM) and voltage-gated calcium channels (Ca(v)s) are crucial for Ca(v) activity-dependent feedback modulation. We recently reported an X-ray structure that shows two Ca(2+)/CaM molecules bound to the Ca(v)1.2 C terminal tail, one at the PreIQ region and one at the IQ domain. Surprisingly, the asymmetric unit of the crystal showed a dimer in which Ca(2+)/CaM bridged two PreIQ helixes to form a 4:2 Ca(2+)/CaM:Ca(v) C-terminal tail assembly. Contrary to previous proposals based on a similar crystallographic dimer, extensive biochemical analysis together with subunit counting experiments of full-length channels in live cell membranes failed to find evidence for multimers that would be compatible with the 4:2 crossbridged complex. Here, we examine this possibility further. We find that CaM over-expression has no functional effect on Ca(v)1.2 inactivation or on the stoichiometry of full-length Ca(v)1.2. These data provide further support for the monomeric Ca(v)1.2 stoichiometry. Analysis of the electrostatic surfaces of the 2:1 Ca(2+)/CaM:Ca(V) C-terminal tail assembly reveals notable patches of electronegativity. These could influence various forms of channel modulation by interacting with positively charged elements from other intracellular channel domains.     

Calmodulin overexpression does not alter Cav1.2 function or oligomerization state

Interactions between calmodulin (CaM) and voltage-gated calcium channels (Ca_vs) are crucial for Ca_v activity-dependent feedback modulation. We recently reported an X-ray structure that shows two Ca²⁺/CaM molecules bound to the Ca_v1.2 C terminal tail, one at the PreIQ region and one at the IQ domain. Surprisingly, the asymmetric unit of the crystal showed a dimer in which Ca²⁺/CaM bridged two PreIQ helixes to form a 4:2 Ca²⁺/CaM:Ca_v C-terminal tail assembly. Contrary to previous proposals based on a similar crystallographic dimer, extensive biochemical analysis together with subunit counting experiments of full-length channels in live cell membranes failed to find evidence for multimers that would be compatible with the 4:2 crossbridged complex. Here, we examine this possibility further. We find that CaM over-expression has no functional effect on Ca_v1.2 inactivation or on the stoichiometry of full-length Ca_v1.2. These data provide further support for the monomeric Ca_v1.2 stoichiometry. Analysis of the electrostatic surfaces of the 2:1 Ca²⁺/CaM:CaV C-terminal tail assembly reveals notable patches of electronegativity. These could influence various forms of channel modulation by interacting with positively charged elements from other intracellular channel domains.     

Using a stick does not necessarily alter judged distances or reachability

Background

It has been reported that participants judge an object to be closer after a stick has been used to touch it than after touching it with the hand. In this study we try to find out why this is so.

Methodology

We showed six participants a cylindrical object on a table. On separate trials (randomly intermixed) participants either estimated verbally how far the object is from their body or they touched a remembered location. Touching was done either with the hand or with a stick (in separate blocks). In three different sessions, participants touched either the object location or the location halfway to the object location. Verbal judgments were given either in centimeters or in terms of whether the object would be reachable with the hand. No differences in verbal distance judgments or touching responses were found between the blocks in which the stick or the hand was used.

Conclusion

Instead of finding out why the judged distance changes when using a tool, we found that using a stick does not necessarily alter judged distances or judgments about the reachability of objects.     

Light level does not alter ethylene sensitivity in radish or pea

Ethylene accumulation occurs in many plant growth environments. In some instances, low photosynthetic photon flux (PPF) is also a stress factor. Ethylene helps regulate the shade-avoidance mechanism and synthesis rates can be altered by light. We thus hypothesized that ethylene sensitivity in whole plants may be altered in low light. Radish (Raphanus sativus) and pea (Pisum sativum) plants were selected as models due to their rapid growth, use in previous studies and difference in growth habit. We first characterized radish and pea sensitivity to ethylene. Radish vegetation was less sensitive to ethylene than pea vegetation. Pea reproductive yield was highly sensitive. Plants grown under low light levels are typically etiolated and less robust than plants grown under higher light. In a second series of studies we examined the interaction of ethylene (50 ppb pea, 200 ppb radish) with PPFs from 50 to 400 μmol m^?2 s^?1. There was no statistically significant interaction between ethylene sensitivity and PPF, indicating that high PPF does not mitigate the detrimental effects of chronic low-level ethylene exposure. This also suggests there is no crosstalk between the shade avoidance pathway and the primary ethylene signaling pathway.     

Ghrelin increases the motivation to eat, but does not alter food palatability

Homeostatic eating cannot explain overconsumption of food and pathological weight gain. A more likely factor promoting excessive eating is food reward and its representation in the central nervous system (CNS). The anorectic hormones leptin and insulin reduce food reward and inhibit related CNS reward pathways. Conversely, the orexigenic gastrointestinal hormone ghrelin activates both homeostatic and reward-related neurocircuits. The current studies were conducted to identify in rats the effects of intracerebroventricular ghrelin infusions on two distinct aspects of food reward: hedonic valuation (i.e., "liking") and the motivation to self-administer (i.e., "wanting") food. To assess hedonic valuation of liquid food, lick motor patterns were recorded using lickometry. Although ghrelin administration increased energy intake, it did not alter the avidity of licking (initial lick rates or lick-cluster size). Several positive-control conditions ruled out lick-rate ceiling effects. Similarly, when the liquid diet was hedonically devalued with quinine supplementation, ghrelin failed to reverse the quinine-associated reduction of energy intake and avidity of licking. The effects of ghrelin on rats' motivation to eat were assessed using lever pressing to self-administer food in a progressive-ratio paradigm. Ghrelin markedly increased motivation to eat, to levels comparable to or greater than those seen following 24 h of food deprivation. Pretreatment with the dopamine D1 receptor antagonist SCH-23390 eliminated ghrelin-induced increases in lever pressing, without compromising generalized licking motor control, indicating a role for D1 signaling in ghrelin's motivational feeding effects. These results indicate that ghrelin increases the motivation to eat via D1 receptor-dependent mechanisms, without affecting perceived food palatability.     

Amyloid beta peptide ratio 42/40 but not A beta 42 correlates with phospho-Tau in patients with low- and high-CSF A beta 40 load

Neurochemical dementia diagnostics (NDD) can significantly improve the clinically based categorization of patients with early dementia disorders, and the cerebrospinal fluid (CSF) concentrations of amyloid beta peptides ending at the amino acid position of 42 (A beta x-42 and A beta 1-42) are widely accepted biomarkers of Alzheimer's disease (AD). However, in subjects with constitutively high- or low-CSF concentrations of total A beta peptides (tA beta), the NDD interpretation might lead to erroneous conclusions as these biomarkers seem to correlate better with the total A beta load than with the pathological status of a given patient in such cases. In this multicenter study, we found significantly increased CSF concentrations of phosphorylated Tau (pTau181) and total Tau in the group of subjects with high CSF A beta x-40 concentrations and decreased A beta x-42/x-40 concentration ratio compared with the group of subjects with low CSF A beta x-40 and normal A beta ratio (p<0.001 in both cases). Furthermore, we observed significantly decreased A beta ratio (p<0.01) in the group of subjects with APOE epsilon 4 allele compared with the group of subjects without this allele. Surprisingly, patients with low-A beta x-40 and the decreased A beta ratio characterized with decreased pTau181 (p<0.05), and unaltered total Tau compared with the subjects with high A beta x-40 and the A beta ratio in the normal range. We conclude that the amyloid beta concentration ratio should replace the 'raw' concentrations of corresponding A beta peptides to improve reliability of the neurochemical dementia diagnosis.     

6-hydroxydopamine does not reduce molecular oxygen directly, but requires a coreductant

The autoxidation of 6-hydroxydopamine (6HODA) was virtually blocked (k2 less than 10(-15) M-1 S-1 at pH 8.0, ionic strength 0.04) by the simultaneous presence of diethylenetriaminepentaacetic acid (DTPA), catalase, and superoxide dismutase (SOD). No quinone product or oxygen consumption was detectable after 12 min under these conditions. Thus, if 6HODA is to react with molecular oxygen at a measurable rate, some other redox species is required as a coreductant. The subsequent addition of formate or mannitol proved capable of overcoming the total inhibition induced by the mixture of catalase, SOD, and DTPA. The simplest interpretation of the data is that most of the autoxidation of 6HODA, as commonly observed, involves successive reduction of a series of metal-bound species of oxygen; the actual transfer of electrons occurring within a ternary reductant-metal-oxygen transition state.     

Cyclosporin A inhibits the production of gamma interferon (IFN gamma), but does not inhibit production of virus-induced IFN alpha/beta

The effect of cyclosporin A (CsA) on the production of gamma interferon (IFN gamma) versus IFN alpha/beta was studied using mouse and human lymphocytes and fibroblasts. Spleen cells from C57Bl/6 mice produced low but significant levels (40-60 U/ml) of IFN gamma after 2 to 3 days of culture with irradiated DBA spleen cells. The addition of CsA at concentrations as low as 0.1 microgram/ml completely inhibited (less than 10 U/ml) IFN gamma production in these cultures. High levels of IFN gamma (170-1200 U/ml) were produced when either C57Bl/6 spleen cells or Ficoll-Hypaque-purified human peripheral blood lymphocytes (PBL) were cultured with the T-cell mitogen staphylococcal enterotoxin A (SEA). The addition of CsA (0.1 microgram/ml) to these cultures also completely inhibited (less than 10 U/ml) IFN gamma production. This inhibition was shown not to be due to a change in the kinetics of IFN gamma production or to a change in the amount of SEA required for stimulation. IFN gamma production in SEA-stimulated mouse spleen cells was inhibited at 3 days of culture even when CsA was added at 24 or 48 hr postculture initiation. Thus, CsA inhibits IFN gamma production even when early events associated with lymphocyte activation have been allowed to take place. In contrast to IFN gamma production, IFN alpha/beta production by Newcastle disease virus (NDV)-infected mouse and human lymphocytes or fibroblasts was not inhibited by the addition of CsA (1 microgram/ml). CsA also did not block the action of IFN gamma or IFN alpha/beta since addition of CsA (1 microgram/ml) to reference IFN standards had no effect on their antiviral activity. Thus, CsA inhibits the production of IFN gamma by T cells but appears to have no effect on the production of IFN alpha/beta by virus-infected cells or on the antiviral action of already produced IFN gamma and IFN alpha/beta.     

Hypoglycaemia downregulates endotoxin-induced production of tumour necrosis factor-alpha,but does not affect IL-1beta,IL-6, or IL-10

The aim of the present study was to investigate the effect of hypoglycaemia on the production capacity of the proinflammatory cytokines tumour necrosis factor-alpha (TNFalpha) and interleukin-1beta (IL-1beta) in subjects with and without diabetes. Hyperinsulinaemic (360 pmolm(-2) x min(-1)) stepped hypoglycaemic (5.0-3.5-2.5 mmoll(-1)) glucose clamps were performed in eight diabetic patients and in six non-diabetic subjects, and hyperinsulinaemic normoglycaemia (5.0 mmoll(-1)) control experiments were performed in four non-diabetic subjects. Circulating levels of cytokines and endotoxin-induced production of TNFalpha, IL-1beta, IL-6, and IL-10 were assessed. The effects of insulin and adrenaline were measured in separate in vitro experiments. In non-diabetic subjects, hypoglycaemia downregulated the production capacity of TNFalpha in a concentration-dependent fashion (P=0.007), but not of IL-1beta, IL-6, or IL-10. Compared to controls, the production capacity of TNFalpha in diabetic patients was already suppressed at normoglycaemia (P=0.02) and only fell in response to hypoglycaemic nadir (P=0.04). The downregulation of TNFalpha could not be explained by increased insulin or adrenaline levels. We conclude that hypoglycaemia specifically downregulates TNFalpha production capacity. Diabetic patients already have a suppressed TNFalpha production capacity at non-hypoglycaemic levels.