Purification and characterization of aspartic protease derived from Sf9 insect cells

An aspartic protease that is significantly produced by baculovirus-infected Spodoptera frugiperda Sf9 insect cells was purified to homogeneity from a growth medium. To monitor aspartic protease activity, an internally quenched fluoresce (IQF) substrate specific to cathepsin D was used. The purified aspartic protease showed a single protein band on SDS-PAGE with an apparent molecular mass of 40 kDa. The N-terminal amino acid sequence of the enzyme had a high homology to a Bombyx mori aspartic protease. The enzyme showed greatest affinity for the IQF substrate at pH 3.0 with a K(m) of 0.85 μM. The k(cat) and k(cat)/K(m) values were 13 s(-1) and 15 s(-1) μM(-1) respectively. Pepstatin A proved to be a potent competitive inhibitor with inhibitor constant, K(i), of 25 pM.     

Expression of GD2 and GD3 gangliosides in human embryonic neural stem cells

NSCs (neural stem cells) are undifferentiated neural cells endowed with a high potential for proliferation and a capacity for self-renewal with retention of multipotency to differentiate into neurons and glial cells. It has been recently reported that GD3, a b-series ganglioside, is a marker molecule for identifying and isolating mouse NSCs. However, the expression of gangliosides in human NSCs is largely unknown. In the present study, we analysed the expression of gangliosides, GD2 and GD3, in human NSCs that were isolated from human brains at gestational week 17 in the form of neurospheres, which are floating clonal aggregates formed by NSCs in vitro. Employing immunocytochemistry, we found that human NSCs were strongly reactive to anti-GD2 antibody and relatively weakly reactive to anti-GD3 antibody. Treatment of these cells with an organic solvent such as 100% methanol, which selectively removes glycolipids from plasma membrane, abolished the immunoreactivity with those antibodies, indicating that the reactivity was due to GD2 and GD3, but not to GD2-/GD3-like glycoproteins or proteoglycans. The immunoreactivity of human NSCs to antibody against SSEA-1 (stage-specific embryonic antigen-1), a well-known carbohydrate antigen of NSCs, was not decreased by the treatment with 100% methanol, indicating that SSEA-1 is mainly carried by glycoproteins and/or proteoglycans in human NSCs. Our study suggests that GD2 and GD3 can be marker gangliosides for identifying human NSCs.     

Cytokine and Nitric Oxide Levels in Patients with Sepsis – Temporal Evolvement and Relation to Platelet Mitochondrial Respiratory Function

Background

The levels of nitric oxide (NO) and various cytokines are known to be increased during sepsis. These signaling molecules could potentially act as regulators and underlie the enhancement of mitochondrial function described in the later phase of sepsis. Therefore, we investigated the correlation between observed changes in platelet mitochondrial respiration and a set of pro- and anti-inflammatory cytokines as well as NO plasma levels in patients with sepsis.

Methods and Results

Platelet mitochondrial respiration and levels of TNFα, MCP-1 (monocyte chemotactic protein-1), INFγ (interferon-γ), IL-1β, IL-4, IL-5, IL-6, IL-8, IL-10 and IL-17 and NO were analyzed in 38 patients with severe sepsis or septic shock at three time points during one week following admission to the ICU. Citrate synthase, mitochondrial DNA and cytochrome c were measured as markers of cellular mitochondrial content. All mitochondrial respiratory states increased over the week analyzed (p<0.001). IL-8 levels correlated with maximal mitochondrial respiration on day 6–7 (p = 0.02, r² = 0.22) and was also higher in non-survivors compared to survivors on day 3–4 and day 6–7 (p = 0.03 respectively). Neither NO nor any of the other cytokines measured correlated with respiration or mortality. Cytochrome c levels were decreased at day 1–2 by 24±5% (p = 0.03) and returned towards values of the controls at the last two time points. Citrate synthase activity and mitochondrial DNA levels were similar to controls and remained constant throughout the week.

Conclusions

Out of ten analyzed cytokines and nitric oxide, IL-8 correlated with the observed increase in mitochondrial respiration. This suggests that cytokines as well as NO do not play a prominent role in the regulation of platelet mitochondrial respiration in sepsis. Further, the respiratory increase was not accompanied by an increase in markers of mitochondrial content, suggesting a possible role for post-translational enhancement of mitochondrial respiration rather than augmented mitochondrial mass.     

Extracting Objective Estimates of Sedentary Behavior from Accelerometer Data: Measurement Considerations for Surveillance and Research Applications

BackgroundAccelerometer-based activity monitors are widely used in research and surveillance applications for quantifying sedentary behavior (SB) and physical activity (PA). Considerable research has been done to refine methods for assessing PA, but relatively little attention has been given to operationalizing SB parameters (i.e., sedentary time and breaks) from accelerometer data - particularly in relation to health outcomes. This study investigated: (a) the accrued patterns of sedentary time and breaks; and (b) the associations of sedentary time and breaks in different bout durations with cardiovascular risk factors.MethodsAccelerometer data on 5,917 adults from the National Health Examination and Nutrition Survey (NHANES) 2003–2006 were used. Sedentary time and breaks at different bout durations (i.e., 1, 2–4, 5–9, 10–14, 15–19, 20–24, 25–29, and ≥30-min) were obtained using a threshold of <100 counts per minute. Sedentary time and breaks were regressed on cardiovascular risk factors (waist circumference, triglyceride, and high-density lipoprotein cholesterol) and body mass index across bout durations.ResultsThe results revealed that the majority of sedentary time occurred within relatively short bout durations (≈70% and ≈85% for <5-min and <10-min, respectively). The associations of sedentary time and breaks with health outcomes varied depending on how bout time was defined. Estimates of SB parameters based on bout durations of 5 min or shorter were associated with reduced cardiovascular risk factors while durations longer than 10-min were generally associated with increased risk factors.ConclusionsThe present study demonstrates that the duration of sedentary bouts should be further considered when operationalizing the SB parameters from accelerometer data. The threshold of 5 minutes to define a bout is defensible, but a 10 minute threshold would provide a more conservative estimate to clearly capture the prolonged nature of sedentary behavior. Additional research is needed to determine the relative sensitivity and specificity of these thresholds.     

Gravity-Induced Modifications to Development in Hypocotyls of Arabidopsis Tubulin Mutants

We investigated the roles of cortical microtubules in gravity-induced modifications to the development of stem organs by analyzing morphology and orientation of cortical microtubule arrays in hypocotyls of Arabidopsis (Arabidopsis thaliana) tubulin mutants, tua3(D205N), tua4(S178Δ), and tua6(A281T), cultivated under 1g and hypergravity (300g) conditions. Hypocotyls of tubulin mutants were shorter and thicker than the wild type even at 1g, and hypergravity further suppressed elongation and stimulated expansion. The degree of such changes was clearly smaller in tubulin mutants, in particular in tua6. Hypocotyls of tubulin mutants also showed either left-handed or right-handed helical growth at 1g, and the degree of twisting phenotype was intensified under hypergravity conditions, especially in tua6. Hypergravity induced reorientation of cortical microtubules from transverse to longitudinal directions in epidermal cells of wild-type hypocotyls. In tubulin mutants, especially in tua6, the percentage of cells with longitudinal microtubules was high even at 1g, and it was further increased by hypergravity. The twisting phenotype was most obvious at cells 10 to 12 from the top, where reorientation of cortical microtubules from transverse to longitudinal directions occurred. Moreover, the left-handed helical growth mutants (tua3 and tua4) had right-handed microtubule arrays, whereas the right-handed mutant (tua6) had left-handed arrays. There was a close correlation between the alignment angle of epidermal cell files and the alignment of cortical microtubules. Gadolinium ions, blockers of mechanosensitive ion channels (mechanoreceptors), suppressed the twisting phenotype in tubulin mutants under both 1g and 300g conditions. Microtubule arrays in tubulin mutants were oriented more transversely by gadolinium treatment, irrespective of gravity conditions. These results support the hypothesis that cortical microtubules play an essential role in maintenance of normal growth phenotype against the gravitational force, and suggest that mechanoreceptors are involved in modifications to morphology and orientation of microtubule arrays by 1g gravity and hypergravity in tubulin mutants.The direction of cell expansion is important for determining the shape of whole plant body. Cortical microtubules are assumed to be responsible for anisotropic expansion of plant cells (Wasteneys and Galway, 2003; Lloyd and Chan, 2004; Mathur, 2004; Baskin, 2005; Paredez et al., 2008). The prevailing view is that cortical microtubule arrays direct or constrain the movement of the cellulose synthase complexes and thus align nascent cellulose microfibrils in the same direction in the innermost layer of the cell wall (Baskin, 2001), although some other mechanisms may also be involved (Baskin, 2001; Sugimoto et al., 2003; Wasteneys, 2004).It is evident that orientation of cortical microtubules plays an essential role in creating the distinct shape of higher plant organs, even if there is uncertainty over the mechanism by which microtubules influence morphogenesis. The importance of cortical microtubule arrays for anisotropic growth has been documented by pharmacological studies and experiments with helical growth mutants of Arabidopsis (Arabidopsis thaliana). Mutants on α- and β-tubulins as well as microtubule-associated proteins show either left-handed or right-handed helical growth (Thitamadee et al., 2002; Nakajima et al., 2004; Sedbrook et al., 2004; Shoji et al., 2004). The rapidly elongating cells of these mutants skew consistently either to the right or to the left and exhibit cortical microtubule arrays that form shallow helices with fixed handedness (Thitamadee et al., 2002; Abe and Hashimoto, 2005; Ishida et al., 2007). Cortical microtubule arrays in the left-handed helical growth mutants form right-handed helix, whereas those in right-handed helical growth mutants form left-handed helix (Thitamadee et al., 2002; Abe and Hashimoto, 2005; Ishida et al., 2007). These results indicate that dysfunctional cortical microtubules are arranged in helical arrays and affect the direction of cell expansion.The gravitational force is one of the environmental factors that determine the plant body shape. Under hypergravity conditions produced by centrifugation, plants generally have a shorter and thicker body (Soga et al., 2006). Namely, hypergravity modifies growth anisotropy. In Arabidopsis hypocotyls, the expression of most α- and β-tubulin genes was up-regulated by hypergravity (Yoshioka et al., 2003; Matsumoto et al., 2007). In protoplasts of Brassica hypocotyls, hypergravity stimulated the regeneration of cortical microtubules into parallel arrays (Skagen and Iversen, 1999), and in azuki bean (Vigna angularis) epicotyls it increased the percentage of cells with longitudinal cortical microtubules (Soga et al., 2006). The reorientation of cortical microtubules from transverse to longitudinal directions may be involved in modifications by hypergravity to growth anisotropy.The aim of this study was to clarify the roles of cortical microtubules in gravity-induced modifications to development of stem organs. For this purpose, we examined the changes in growth, morphology, and orientation of cortical microtubule arrays in hypocotyls of Arabidopsis amino acid substitution mutants in α-tubulin structure, tua3, tua4, and tua6, grown under 1g and 300g conditions. We have reported the possible involvement of mechanosensitive ion channels (mechanoreceptors) in hypergravity-induced modifications to growth and cell wall properties (Soga et al., 2004, 2005, 2006). Thus, we also examined the effect of blockers of mechanoreceptors on helical growth and orientation of cortical microtubule arrays in the tubulin mutants.     

Characterization of a bicistronic knock-in reporter mouse model for investigating the role of CABLES2 in vivo

Two members of the CDK5 and ABL enzyme substrate (CABLES) family, CABLES1 and CABLES2, share a highly homologous C-terminus. They interact and associate with cyclin-dependent kinase 3 (CDK3), CDK5, and c-ABL. CABLES1 mediates tumor suppression, regulates cell proliferation, and prevents protein degradation. Although Cables2 is ubiquitously expressed in adult mouse tissues at RNA level, the role of CABLES2 in vivo remains unknown. Here, we generated bicistronic Cables2 knock-in reporter mice that expressed CABLES2 tagged with 3×FLAG and 2A-mediated fluorescent reporter tdTomato. Cables2-3×FLAG-2A-tdTomato (Cables2^Tom) mice confirmed the expression of Cables2 in various mouse tissues. Interestingly, high intensity of tdTomato fluorescence was observed in the brain, testis and ovary, especially in the corpus luteum. Furthermore, immunoprecipitation analysis using the brain and testis in Cables2^Tom/Tom revealed interaction of CABLES2 with CDK5. Collectively, our new Cables2 knock-in reporter model will enable the comprehensive analysis of in vivo CABLES2 function.     

JM-1232(−) and propofol,a new combination of hypnotics with short-acting and non-cumulative preferable properties

Drug interactions are significant in anesthesiology because drug combinations can potentially possess novel properties. The pharmacological advantages of a new combination of the benzodiazepine receptor agonist JM-1232(−) and propofol were investigated in mice. Male adult mice were administered JM-1232(−) or propofol or combinations of the two drugs intravenously. Loss of the righting reflex was evaluated as achieving hypnosis, and the time until recovery of the reflex was measured as hypnosis time. After determining the ED₅₀, doses double and triple the ED₅₀ of propofol were injected with JM-1232(−) to compare hypnosis time. The injections were repeated four times, and the hypnosis times were compared. Flumazenil was administered separately immediately after the last dose was injected. The ED₅₀ values ([95% confidence interval]) for hypnosis were 3.76 [3.36–4.10] for JM-1232(−) and 9.88 [8.03–11.58] mg kg⁻¹ for propofol. Co-administration of 0.5 and 1 mg kg⁻¹ JM-1232(−) reduced the ED₅₀ values of propofol to 1.76 [1.21–2.51] and 1.00 [0.46–1.86] mg kg⁻¹, respectively. The drug combination for hypnosis produced a supra-additive interaction. Hypnosis time was significantly shorter in the groups given the mixtures compared to each hypnotic administered alone. After repeated injections, hypnosis time with the mixtures showed smaller prolongation than that with the hypnotic alone. Flumazenil completely restored the recovery time after anesthesia. The combination of JM-1232(−) and propofol showed a supra-additive interaction, and the reduced hypnotic dose contributed to a faster recovery even after multiple injections.     

Change of Cu,Zn-superoxide dismutase activity of guinea pig lung in experimental asthma

Correlation between the level of reactive oxygen species (ROS) generated by airway inflammatory cells and superoxide dismutase (SOD) activity of pulmonary tissue during an asthma attach was investigated in a guinea pig model of allergic asthma. In addition, the influence of SOD inhibition by diethyldithiocarbamate (DDC, Cu-chelating agent) on the airway was investigated in terms of pulmonary function during an asthma attach. Relative to controls, the capacity of bronchoalveolar lavage fluid (BAL) cells to release ROS was significantly increased in guinea pigs sensitized with ovalbumin (OA) as the antigen, and significantly increased in guinea pigs with an asthma attack provoked by the inhalation of OA. SOD activity was increased significantly in the antigen-sensitized group. The asthma provocation group showed a tendency for increase in total SOD activity, compared with the sensitization group, whose increase was dependent on the increase in copper, zinc-SOD (Cu, Zn-SOD) activity. Pretreatment with DDC increased the severity and duration of the asthma attack. These results were indicated that Cu, Zn-SOD was closely involved in the asthma process, particularly in the scavenging of oxygen radicals secreted from BAL cells.     

Serotonin differentially regulates short- and long-term prediction of rewards in the ventral and dorsal striatum

Background

The ability to select an action by considering both delays and amount of reward outcome is critical for maximizing long-term benefits. Although previous animal experiments on impulsivity have suggested a role of serotonin in behaviors requiring prediction of delayed rewards, the underlying neural mechanism is unclear.

Methodology/Principal Findings

To elucidate the role of serotonin in the evaluation of delayed rewards, we performed a functional brain imaging experiment in which subjects chose small-immediate or large-delayed liquid rewards under dietary regulation of tryptophan, a precursor of serotonin. A model-based analysis revealed that the activity of the ventral part of the striatum was correlated with reward prediction at shorter time scales, and this correlated activity was stronger at low serotonin levels. By contrast, the activity of the dorsal part of the striatum was correlated with reward prediction at longer time scales, and this correlated activity was stronger at high serotonin levels.

Conclusions/Significance

Our results suggest that serotonin controls the time scale of reward prediction by differentially regulating activities within the striatum.