65 and 66

Overall, the APOE4 mice spent more time in the open arms than their APOE3 counterpart, results that are in contradiction with previous studies. 65 Furthermore, supplementation with vitamins influenced in a genotype-dependent manner the behavior of the mice on this task. In the APOE4 mice, the supplementation with antioxidants increased anxiety. This was in definite contrast with several studies relating that vitamin E depletion increases anxiety. 67 Furthermore, other studies have demonstrated a decrease in anxiety in rats supplemented with vitamins E and C. 68 In our active avoidance paradigm, the results differed depending on whether we analyzed the discriminative component or the avoidance component of the task. In the discriminative component, Enzalutamide in vivo which is the component of active avoidance that the mice learned first, there was a definite improvement in performance following exercise and antioxidant treatment in the APOE3 mice. The lack of significant improvement in the APOE4 mice could be due to a maximum plateau of performance due to the set criterion. The criterion was set as the number of trials to reach four out of five correct turns, therefore four trials would be the minimum number of trials than a mouse

could take. On average the SedCon APOE4 mice took between six and eight trials, thereby making it difficult to detect a significant effect of Treatment. The effects of Treatment were mostly due to exercise Treatment as the performance of the APOE3 mice remained largely Autophagy inhibitors unaffected by supplementation with antioxidants. In the avoidance component of the task, exercise training improved performance of the APOE mice, irrespective of genotype

in the acquisition phase. Interestingly, supplementation with antioxidants was only effective in the APOE4 mice. This is most likely due to the transporter protein being dysfunctional 69 and APOE4 mice having lower vitamin E levels, 70 therefore more responsive to antioxidant supplementation. Physical activity has been shown to reduce AD risk, 41, 42 and 43 to improve cognitive function and to have a positive impact on functional plasticity. 44 Interestingly, APOE4 allele carriers Liothyronine Sodium with a sedentary life style have been shown to be more vulnerable to excessive amyloid deposition in brain. 45 and 71 Physical activity levels have been strongly positively associated with cognitive function in individuals carrying APOE4 72 and 73 supported by transgenic mouse model carrying human APOE4. 33 These studies focused on individuals or mice in which cognitive dysfunction was present, while our study demonstrated that improvement can also be attained without apparent cognitive dysfunction and did not seem to be dependent upon the APOE genotype. Studies on combination of antioxidant and exercise have led to conflicting results.

Previous studies suggested that a glutamatergic-purinergic signaling pathway prevents hypoosmotic swelling of Müller cells in the rodent retina selleck chemicals llc by vesicular release of glutamate (Wurm et al., 2008 and Wurm

et al., 2010; Figure 4A). Therefore, we tested whether this pathway was defect in BoNT/B-expressing Müller cells. Müller cells from Tam-injected bigenic mice, which expressed the BoNT/B transgene as indicated by the presence of EGFP (Figure 4B), had similar cross sectional areas as cells from Tam-injected monogenic mice (Figure 4C). However, these cells swelled in hypotonic solution, while Müller cells from monogenic animals maintained their cell volume (Figure 4D) indicating a toxin-induced defect in volume regulation. If the Vorinostat nmr swelling of toxin-expressing Müller cells was due to the block of glutamate release, coapplication of glutamate should prevent the swelling of these cells. As shown in Figure 4D, this

was indeed the case thus confirming the involvement of exocytotic glutamate release in volume regulation. Vascular endothelial growth factor (VEGF), which activates the volume-regulating cascade upstream of glutamate release (Wurm et al., 2008), failed to abolish swelling of Müller cells from bigenic mice (Figure 4D), whereas it prevented hypotonic swelling of Müller cells from monogenic mice due to barium-induced block of inwardly rectifying potassium channels (Wurm et al., 2008) (Figure 4D). Toxin expression in Müller cells may have provoked reactive gliosis and thereby perturbed glial volume regulation (Pannicke et al., 2004, Pannicke et al.,

2005 and Sene et al., 2009). However, levels of glial fibrillary acidic protein (GFAP) were similar in retinae from Tam-injected mono- and bigenic mice (data not shown). Additional hallmarks of Müller cell reactivity are a downregulation of potassium inward currents and an increase in membrane capacitance (Pannicke et al., 2005). Our patch-clamp recordings revealed comparable amplitudes of inward currents and even a significant (p < 0.02; Student's t test) decrease in membrane capacitance in acutely isolated Müller cells from Tam-injected bigenic (2.4 ± 0.7 nA; 40 ± 12 pF; n = 33 cells from 8 mice) compared to monogenic mice (2.2 ± 0.6 nA; 49 ± 12 pF; most n = 29 cells, 7 mice). The VEGF-induced glutamate release from Müller cells also induces swelling of neurons in the ganglion cell layer independently from a hypo-osmotic challenge (Wurm et al., 2008). To test whether this effect was eliminated by glial toxin expression, we measured the size of neuronal somata that were close to endfeet of toxin- and EGFP-expressing Müller cells from Tam-injected bigenic mice (Figure 4E). Indeed, these neurons did not show VEGF-induced swelling, whereas the effect was present in cells from Tam-injected monogenic animals (Figure 4E, F). As for Müller cells, the mean cross sectional area of untreated neurons was similar in mono- and bigenic mice (Figure 4C).

The characteristics of all OSI-906 datasheet vaccines have been previously reported [10], [11] and [12]. Both studies were conducted in accordance with the Code of Ethics of the World Medical Association

(Declaration of Helsinki). Parents or guardians recorded daily temperatures and signs or symptoms of respiratory illness and were instructed to promptly notify study personnel if their child developed qualifying symptoms. They were also contacted every 7–10 days throughout the influenza season. Nasal swabs were collected if a child had ≥1 of the following: acute otitis media (suspected or diagnosed), fever, pneumonia, pulmonary congestion, shortness of breath, or wheezing, or ≥2 of the following symptoms concurrently: chills, cough, decreased activity, headache, irritability, muscle aches, pharyngitis, rhinorrhea, or vomiting. Central laboratories evaluated nasal swabs for the presence of influenza virus by viral culture; wild-type serotypes were identified using antigenic methods. Laboratory-confirmed cases of influenza were classified as moderate/severe influenza if there was any documentation

of fever >39 °C, acute otitis media, or lower respiratory tract illness (defined as healthcare provider-confirmed shortness of breath, pulmonary congestion, pneumonia, bronchiolitis, bronchitis, wheezing, or croup). All other cases were classified as milder influenza. All children ≥24 months of age were retained in this post hoc analysis. MEK inhibitor cancer Efficacy was calculated as one minus the relative risk of laboratory-confirmed influenza regardless of antigenic match with LAIV versus placebo or IIV. Efficacy was evaluated first against moderate/severe cases of influenza in all children, then against mild cases of influenza only. The 95% CIs of the vaccine efficacy point estimates were obtained by a log-binomial regression. Results

from the two studies were not combined because study 1 assessed LAIV efficacy versus placebo, whereas study 2 assessed LAIV efficacy versus IIV. A total of 1330 children ≥24 months of age in year 1 (LAIV, n = 897; placebo, n = 433) and 1358 children in year 2 (LAIV, n = 917; placebo, n = 441) were enrolled in study 1. The attack rates of moderate/severe influenza no were 0.6% (5/897) in year 1 and 1.1% (10/917) in year 2 in the LAIV group versus 12.0% (52/433) in year 1 and 9.5% (42/441) in year 2 in the placebo group, resulting in efficacy estimates of 95.4% (95% CI: 88.5, 98.1) in year 1 and 88.5% (77.4, 94.9) in year 2 ( Figs. 1A and 1B). The attack rates of mild influenza were 0.6% (5/892) in year 1 and 0.6% (5/907) in year 2 in the LAIV group versus 6.6% (25/381) in year 1 and 3.6% (14/399) in year 2 in the placebo group, resulting in efficacy estimates of 91.4% (77.9, 96.7) and 84.2% (56.7, 94.3) in year 1 and year 2, respectively ( Figs. 1A and 1B). In year 1, both A/H3N2 and B strains circulated. Efficacy against moderate/severe influenza for A/H3N2 and B strains was 95.7% (86.5, 99.2) and 95.8% (83.0, 99.

There are few rigorous 31P-MRS studies of healthy young people but current data indicate that age- and sex-related differences in muscle metabolism are dependent on the intensity of the imposed exercise. During moderate intensity exercise no age- or sex-related differences in metabolism have been observed but during exercise above the ITPi/PCr the anaerobic energy contribution for a given increase in normalised power has been demonstrated to be lower in children than adults and in boys compared to girls. In females the

increased glycolytic activity has been related to stage of maturation. The lower accumulation of Pi and fall in pH and PCr are consistent with a greater recruitment of type I muscle fibres in children compared to adults Dabrafenib price and in boys compared to girls. The development and application of non-invasive technologies and methodologies such as 31P-MRS and breath-by-breath pV˙O2 kinetics to interrogate muscles in vivo   has enhanced our understanding of paediatric exercise metabolism and provided new insights into data obtained from Cell Cycle inhibitor conventional techniques. Rigorously designed, executed, and interpreted 31P-MRS studies with children are sparse and most studies are limited by small sample sizes but initial research has clearly indicated the huge untapped potential of this technique. 31P-MRS studies are costly and the close relationship

between PCr kinetics and pV˙O2 kinetics encourages the use of more child-friendly and less expensive pV˙O2 kinetics with young people. Appropriate data collection, modelling and analysis techniques using pV˙O2 kinetics with children are now well-established and the recent introduction of the use of experimental models such as priming exercise, work to work transitions, and manipulation

of pedal rates provide intriguing avenues for future research into paediatric exercise metabolism. “
“It is known from animal studies that neuromotor patterns change in respect to gait patterns.1 and 2 Through the use of surface electromyography (EMG), these changes were also investigated in humans. Cell press Based on their earlier study, Hreljac et al.3 have tested the hypothesis that gait pattern was changed from walk to run in order to reduce muscular stress on the dorsiflexor while simultaneously placing more demand on the larger muscles of the lower extremity. EMG activity of the tibialis anterior (TA), medial gastrocnemius (GA), vastus lateralis (VL), biceps femoris long head (BFL), and gluteus maximus (GM) have been monitored while participants walked at constant speeds of 70%, 80%, 90%, and 100% of their preferred walk to run (WR) transition speed and ran at their preferred WR transition speed. Results have shown that the peak normalized EMG activity for TA increased as walking speed increased, then decreased when gait changed to a run at preferred transition speed.

The data originated from a longitudinal study of adolescent health that has been previously described.22 and 23 Briefly, we conducted five waves of adolescent and parent telephone surveys between 2002 and 2009. This study used parent

and adolescent data collected at baseline (2002–2003), wave four (2007–2008), and wave five (2008–2009). We also collected school-level data between October 2007 and February 2008 from high schools attended by adolescent participants. The Dartmouth Committee for the Protection of Human Subjects approved all aspects of this research. In 2002–2003, we surveyed 87% (n = 3705) of students enrolled in grades 4–6 at 26 randomly selected New Hampshire and Vermont public elementary schools. Subsequently, we enrolled 71% (n = 2631) of these students and one of their parents into a longitudinal telephone survey. check details We preferentially surveyed mothers for consistency across waves; if no mother lived in the household, we surveyed the adolescent’s primary caregiver instead. We completed telephone Venetoclax surveys at either wave four or five with 2009 adolescents. Because the majority of adolescents

were enrolled in high school, and athletic programs differed significantly between middle and high schools, we confined our analysis to high school students (n = 1804). If both wave four and five surveys were available, we used whichever survey was conducted closest to the date of school-level data collection (59.4% from wave four). Trained interviewers administered surveys to adolescents and their mothers using a computer assisted telephone interviewing system. Interviewers obtained parent consent and adolescent assent before each survey. In all but a few instances, we surveyed the adolescent before his/her mother. There was no mother Endonuclease or step-mother living in 60 households; in these instances, we surveyed the adolescent’s primary caregiver. The majority of adolescents in our sample tracked

into district-associated catchment high schools. We asked 29 of these high schools to participate in a school-based environmental assessment. Three high schools refused; another three agreed, but never mailed back the written questionnaires. For the 23 participating high schools, athletic directors completed a questionnaire about the school’s athletic program, and physical education (PE) instructors completed a questionnaire about the school’s PE program. Schools were not compensated, but received a summary research report for their participation. From the 1804 high school participants, we further confined our analysis to 1244 adolescents based on the availability of school athletic/PE program data. Our final sample resembled the wave one sample in the percentage of males (49.0% vs. 51.5%), white/Caucasians (91.2% vs. 89.9%), and baseline sports participation (72.5% vs. 68.6%).

Exosomes have been reported to mediate transsynaptic Navitoclax manufacturer protein transfer in Drosophila NMJs ( Korkut et al., 2009), making the possibility that the same mechanism is deployed in the exchange of miRNAs very attractive. Another group of miRNAs involved at the Drosophila larval neuromuscular junction are the miR-310 cluster, miR-310–miR-313, but they appear to be playing an independently presynaptic role not requiring transsynaptic communication.

Loss of the cluster leads to a significant enhancement of neurotransmitter release, which can be rescued with temporally restricted expression of miR-310–miR-313 in larval presynaptic neurons ( Tsurudome et al., 2010). The Kinesin family member Khc-73 is a functional target for the cluster as its expression is increased in cluster mutants and reducing Khc-73 restores normal synaptic function. At later stages of the Drosophila life cycle during periods of tissue remodeling, there is coordinated selleck chemicals llc pre- and postsynaptic expression

of another conserved miRNA, let-7 ( Caygill and Johnston, 2008; Sokol et al., 2008). Loss of the fly let-7 complex (Let-7, miR-100, and miR-125) prevents the normal maturation of these NMJs as these animals metamorphose to adults, largely via regulation of the muscle transcription factor Abrupt. Investigation of miRNA function in many contexts indicates that they often act in concert with transcription factors to augment robustness or mediate feedback in the regulation of effector gene networks (reviewed by Peláez and Carthew, 2012). For example, in the C. elegans neuromuscular system, miR-1 controls both the expression of acetylcholine receptors and the muscle transcription

factor MEF-2 ( Simon et al., 2008). Interestingly, in this model, Phosphoprotein phosphatase MEF-2 is upstream of an unknown transsynaptic retrograde signal that appears to control presynaptic release properties. This miR-1/MEF-2 pathway highlights the intricate ongoing conversation between neurons and their synaptic partners as miR-1 regulates aspects of both pre- and postsynaptic functions at C. elegans neuromuscular junctions. Further exploration of miRNA-transcripton factor interactions in C. elegans has uncovered a role for miRNA in activity-dependent plasticity that is part of normal circuit remodeling during organismal development. In this work, the transcription factor hunchback-like 1 (HBL-1), orthologous to a gene that regulates the timing of neural progenitor fate determination in Drosophila, was found to be specifically expressed in a subset of motor neurons that actively remodel their synaptic connections during larval maturation ( Thompson-Peer et al., 2012). Interestingly, a change in neural activity induced a corresponding change in HBL-1 expression.

, 1994). However, synchrony across large populations of MSNs is rarely seen in healthy individuals and, rather, is a hallmark of striatal dysfunction in motor diseases such as PD and dystonia (Buzsáki et al., 1990, Costa et al., 2006, Gernert et al., 2002, Hammond et al., 2007, Hutchison et al., 2004 and Kühn et al., 2008). In particular, dopamine depletion is associated with increased network oscillations in the β frequency band that may occlude normal signal propagation through the basal ganglia (Brown, 2003,

Kühn et al., 2004 and Mallet et al., 2008b). Although pathological β oscillations after dopamine depletion are a feature of the entire basal ganglia network, some of the most striking shifts in neuronal-firing patterns check details occur in the GP and STN (Bevan et al., 2002, Mallet et al., 2008a and Terman et al.,

2002). These nuclei become highly coupled in an oscillatory pattern after dopamine depletion, and disruption of this abnormal synchrony with deep brain stimulation is an effective therapeutic treatment in patients with PD (Bevan et al., 2002 and Hammond et al., 2007). Although GP neurons do not show a substantial change in average firing rate after dopamine depletion, they do show changes in firing pattern, shifting to a synchronized, bursting mode of firing in resting animals or patients with PD (Brown et al., 2001 and Raz et al., 2000). In part, this altered firing pattern may depend on increased synchronous inhibition from striatal D2 MSNs (Terman et al., 2002). However, a number of other changes in the striatum BAY 73-4506 ic50 have been described after dopamine depletion that could alter the Adenosine output of D2 MSNs. These include

changes in LTD and LTP at excitatory inputs in MSNs (Calabresi et al., 2007, Kreitzer and Malenka, 2008, Lovinger, 2010 and Shen et al., 2008), decreased spine density and loss of glutamatergic synapses onto D2 MSNs (Day et al., 2008), changes in cholinergic signaling (Ding et al., 2006), and changes in a non-FS population of GABAergic interneurons (Dehorter et al., 2009). In this study, we use a simple model of the striatal circuit to demonstrate that experimentally increased innervation of D2 MSNs by FS interneurons may be sufficient to enhance synchrony of D2 MSNs. This, along with other changes in striatal circuitry, could enhance D2 MSN regulation of downstream target neurons and contribute to increased synchrony in the GP and the STN (Burkhardt et al., 2007, Costa et al., 2006, Terman et al., 2002 and Walters et al., 2007). Furthermore, because a subset of GP neurons projects back to striatal interneurons (Bevan et al., 1998 and Gage et al., 2010), this may also amplify indirect-pathway synchrony in the striatum, leading to robust pathological oscillations in the indirect-pathway basal ganglia circuit. Coronal sections containing dorsal striatum were prepared in cold sucrose cutting solution: 79 mM NaCl, 23 mM NaHCO3, 68 mM sucrose, 12 mM glucose, 2.3 mM KCl, 1.1 mM NaH2PO4, 6 mM MgCl2, and 0.5 mM CaCl2.

Internally generated activity may

reflect arousal, attention, anticipation of reward, or other nonsensory signals related to the behavioral state of an organism. How do global brain states alter activity in local cortical networks, and what are the cellular mechanisms responsible for such changes in cortical processing? The most overtly observable brain states are perhaps found in the sleep-wake cycle, with substantial behavioral and perceptual differences between sleeping, drowsy, and alert states. Brain potentials (electroencephalogram; EEG) exhibit prominent slow-wave oscillations (<2 Hz) during natural deep sleep and under anesthesia but not during wakefulness (Steriade et al., 1993b). EEG slow waves derive from relatively click here synchronous discharges of large populations of neurons (Steriade et al., 2001). These discharges are separated by periods of synaptic quiescence, during which virtually all of the thousands of synapses contacting a neuron are inactive. Intracellular Palbociclib purchase recording affords a unique view of network activity, reporting the activity

of these numerous connected cells. The resulting membrane potential (Vm) modulates the impact of subsequent synaptic inputs. In anesthetized animals, Vm at the time of a sensory stimulus strongly influences the amplitude of postsynaptic potentials as well as the number and relative timing of action potentials evoked (Petersen et al., 2003 and Sachdev et al., 2004). In slice, synapses more or less effectively transmit sensory information depending on cortical Vm (Rigas and Castro-Alamancos, 2009 and Watson et al., 2008). Therefore, instantaneous Vm may influence anatomically connected cells’ functional connectivity (Haider and McCormick, 2009) perhaps subserving high-level functions. The temporal patterns of synaptic inputs (network dynamics) during wakefulness are less clear. Heroic sharps recordings initially provided several examples of neurons in multimodal association areas of cat neocortex that exhibit pronounced slow-wave fluctuations

during natural sleep but not wakefulness (Steriade et al., 2001). oxyclozanide Wakefulness was characterized instead by persistent depolarization and high action potential discharge rates. In contrast, a later whole-cell study described low-frequency fluctuations in layer 2/3 pyramidal neurons in rodent primary somatosensory cortex during “quiet wakefulness” (Petersen et al., 2003; see also Poulet and Petersen, 2008), though these have yet to be directly compared to those during sleep/anesthesia. The earlier cat studies observed no slow-wave synaptic patterns during wakefulness, but cell types were unidentified. How arousal affects individual neurons of different types is unresolved. The mechanism by which arousal may alter cortical dynamics is also unclear. Electrical stimulation of the brainstem cholinergic center innervating the thalamus enhances thalamic discharge and tonically depolarizes cortical neurons (Steriade et al.

, 2004). CaMKII and CaN are necessary for attraction and repulsion respectively. Inhibiting CaMKII can block attraction, whereas inhibiting CaN can block repulsion and even convert repulsion to attraction if there are high levels of calcium influx (Wen et al., 2004). Therefore, the ratio of CaMKII to CaN appears to be crucial for determining attraction versus repulsion in guidance responses, CDK inhibitor rather than the absolute activity of each of these molecules. CaMKII and CaN can also regulate activity of one another at different calcium levels through CaN inhibition of the protein inhibitor 1 (I1), an inhibitor of protein phosphatase 1 (PP1), which in turn is an inhibitor of CaMKII (Wen et al.,

2004; Figure 1A). The regulation

of growth cone turning becomes even more complex when one considers other important factors such as the baseline levels of calcium and the activity of cAMP and cGMP. Decreasing the baseline calcium level in the growth cone Selleck AZD2281 converts attraction to repulsion, implying an interaction between the baseline calcium level and the amount of calcium influx in determining the sign of the response (Zheng, 2000). Furthermore, increasing cAMP on one side of the growth cone by presenting an extracellular gradient of cAMP promotes attraction (Lohof et al., 1992 and Murray et al., 2009), whereas lowering the ratio of cAMP to cGMP activity in the presence of a guidance cue gradient can switch turning from attraction to repulsion (Ming et al., 1997, Song et al., 1997, Song et al., 1998 and Nishiyama et al.,

2003). cAMP activates protein kinase A (PKA), which is also known to activate I1 (normally inhibited by CaN), and thus helps to promote attraction by reducing inhibition of CaMKII (Han et al., 2007; Figure 1A). Interpretation of this complex signaling process for guidance must allow for comparison between opposite Phosphatidylinositol diacylglycerol-lyase sides of the growth cone, so that an asymmetric response is possible. Here, we quantitatively test the hypothesis that turning occurs toward the side of the growth cone with the higher CaMKII:CaN ratio, by constructing a mathematical model of the signaling events discussed above. The model is inspired by previous work modeling the analogous switch between long-term potentiation (LTP) and long-term depression (LTD) based on the relative levels of CaMKII and CaN (Lisman, 1989 and Graupner and Brunel, 2007). However, crucially, we consider distinct events occurring on the up-gradient and down-gradient sides of the growth cone, which allows the CaMKII:CaN ratio to be different between the two sides. We first show that this model quantitatively explains the known phenomenology for how calcium and cAMP levels affect the sign of growth cone turning. We then derive predictions from the model for the sign of the response in conditions previously untested experimentally.

We stained the eye discs of the third-instar eyFLP;Rab6 mutant larvae with anti-24B10, anti-Elav, anti-Senseless (labels R8), and anti-Prospero (labels R7). The staining patterns all appear normal, indicating that

there are no differentiation defects in Rab6 mutant eyes (data not shown). We then labeled R7 cells with Pan-R7 Gal4 and assessed the R7 targeting GDC-0068 molecular weight phenotypes of Rab6 mutants. 18.8% ± 3% (n = 265) of the R7 cells in Rab6 mutants (versus 19.7% ± 3% [n = 268] for rich1 mutants) fail to target to the correct layers in the medulla ( Figures 7C, 7C′, and 7D). The similarity in phenotype between Rab6 and rich mutant PR suggests that they function in the same pathway. To test this hypothesis, we removed one copy of Rab6 in the

eyFLP; rich1 or eyFLP; rich2 mutant animals and quantified the R7 targeting defects in the adult medulla. Rab6 heterozygous animals do not have R7 targeting defects, while the hypomorphic allele rich2 only has weak R7 targeting defects (8.5% ± 2%, n = 329; Figures 7F and 7M). We reasoned that loss of one copy of Rab6 in the partial loss of function mutant rich2 would enhance the phenotype. Indeed, loss of a copy of Rab6 enhanced the R7 targeting defects in eyFLP; rich2 animals (17.0% ± 3%, n = 483) similar to the rich1 mutant phenotype (compare Figures 7E and 7F to 7G; Figure 7M). However, loss of a copy of Rab6 does not modify the R7 targeting defects of the null allele rich1 (19.0% ± 3%; n = 308; compare Figures 7E to 7H; Figure 7M). Hence, the phenotypic and genetic data strongly argue this website that rich and Rab6 indeed function in a common pathway. To provide additional evidence that Rich and Rab6 function together,

we overexpressed a constitutively active form of Rab6 (Rab6CA) in rich1 mutant cells using the MARCM system. This greatly suppressed the R7 targeting much defects caused by loss of rich. Moreover, overexpression of a dominant negative form of Rab6 (Rab6DN) did not suppress or enhance the rich1 phenotype, indicating that Rich positively regulates Rab6 activity, possibly as a GEF ( Figures 7I–7L and 7N). To determine if Rab6 physically interacts with Rich, we overexpressed the WD40 domain (AA8–AA521) and RIC1 domain (AA711–AA995) of Rich in S2 cells and performed GST pull-down experiments using GST fusion proteins containing either wild-type Rab6 (Rab6WT), Rab6CA, or Rab6DN. A typical GEF protein tends to bind to the nucleotide-free form (e.g., Rab6WT) or GDP-bound form (e.g., Rab6DN). Consistent with this, both the WD40 domain and the RIC1 domain have a strong affinity for Rab6WT and Rab6DN but not Rab6CA, suggesting that Rich directly regulates Rab6 (Figure 7O). To directly assess the GEF activity of Rich, we overexpressed Rich in S2 cells and performed GTP exchange assay with the cell lysate. In this experimental setting, a positive control, Tiam1, showed proper GTP exchange activity for Rac1.