Turnover of inositol polyphosphate pyrophosphates in pancreatoma cells

There is little information concerning the intracellular function of inositol 1,3,4,5,6-pentakis- and hexakisphosphate, despite their being the most abundant inositol polyphosphates. Current opinions that they play passive roles as antioxidants (Graf, E., Mahoney, J. R., Bryant, R. G., and Eaton, J. W. (1987) J. Biol. Chem. 259, 3620-3624) or "housekeeping" molecules (Berridge, M. J., and Irvine, R. F. (1989) Nature 341, 197-205) arises from belief in their metabolic lethargy. However, we have discovered that cell homogenates, incubated with 5 mM fluoride and 5 mM ATP, converted both inositol hexakisphosphate (Km = 2 +/- 0.5 microM, Vmax = 9 +/- 2 pmol/mg of protein/min) and inositol 1,3,4,5,6-pentakisphosphate (Km = 13 +/- 4 microM, Vmax = 11 +/- 5 pmol/mg of protein/min) to more polar products. These reactions were also observed in intact cells treated with 0.5-20 mM fluoride, and the precursor/product relationships were confirmed by comparing the effects of fluoride on cells differentially labeled with [3H]inositol in either short-term or pulse-chase protocols. The novel products were determined to be inositol pyrophosphates because of their relatively specific hydrolysis by tobacco pyrophosphatase and alkaline phosphatase. The pyrophosphates were metabolized rapidly by cell homogenates back to their pentakisphosphate and hexakisphosphate precursors. This endogenous pyrophosphatase activity was inhibited by up to 99% by 5 mM fluoride in vitro. In intact cells incubated with 10 mM fluoride, about 20% of the inositol 1,3,4,5,6-pentakisphosphate pool, and 50% of the inositol hexakisphosphate pool were each converted to pyrophosphate derivatives within 1 h.     

Role of the cytoskeleton in calcium signaling in NIH 3T3 cells. An intact cytoskeleton is required for agonist-induced [Ca2+]i signaling, but not for capacitative calcium entry

Treatment of NIH 3T3 cells with cytochalasin D (10 microM, 1 h at 37 degrees C) disrupted the actin cytoskeleton and changed the cells from a planar, extended morphology, to a rounded shape. Calcium mobilization by ATP or by platelet-derived growth factor was abolished, while the ability of thapsigargin (2 microM) to empty calcium stores and activate calcium influx was unaffected. Similar experiments with nocodazole to depolymerize the tubulin network yielded identical results. Platelet-derived growth factor induced an increase in inositol phosphates, and this increase was undiminished in the presence of cytochalasin D. Therefore, the blockade of agonist responses by this drug does not result from decreased phospholipase C. Injection of inositol 1,4,5-trisphosphate (IP3) released calcium to the same extent in control and cytochalasin D-treated cells. Confocal microscopic studies revealed a significant rearrangement of the endoplasmic reticulum after cytochalasin D treatment. Thus, disruption of the cytoskeleton blocks agonist-elicited [Ca2+]i mobilization, but this effect does not result from a lower calcium storage capacity, impaired function of the IP3 receptor, or diminished phospholipase C activity. We suggest that cytoskeletal disruption alters the spatial relationship between phospholipase C and IP3 receptors, impairing phospholipase C-dependent calcium signaling. Capacitative calcium entry was not altered under these conditions, indicating that the coupling between depletion of intracellular calcium stores and calcium entry does not depend on a precise structural relationship between intracellular stores and plasma membrane calcium channels.     

Development of a new bubbly-slug interfacial friction model for RELAP5

The development of a new bubbly-slug interfacial friction model for the Pressurized Water Reactor (PWR) safety code RELAP5 is described. The model is based on a set of best-estimate void fraction correlations which cover the full range of geometries and flow conditions encountered in PWR safety analysis. By exploiting the relationship between void fraction and interfacial friction that exists for steady, fully developed flow conditions, the correlations are converted into effective interfacial friction coefficients that can be applied in the code for transient as well as steady-state conditions. Assessments against separate effects tests indicate that the new model is more accurate than the existing model in many situations, particularly rod bundle geometries, and should never be significantly less accurate. The model has been implemented in a local version of RELAP5/MOD2 and in a pre-release version of RELAP5/MOD3 at Idaho National Engineering Laboratory (INEL).     

In vivo characterization of sustained-release formulations of human growth hormone

Long-acting formulations of recombinant human growth hormone (rhGH) were prepared by stabilizing and encapsulating the protein into three different injectable, biodegradable microsphere formulations composed of polymers of lactic and glycolic acid. The formulations were compared in juvenile rhesus monkeys by measuring the serum levels of rhGH and two proteins induced by hGH, insulin-like growth factor-I and IGF binding protein-3 (IGFBP-3) after single s.c. administration. All three formulations, which differed principally in the composition of the polymer, provided sustained elevated levels of all three proteins for several weeks, and the rate of release of rhGH differed among the formulations consistent with the molecular weight of the polymer used. All three formulations induced a higher level of insulin-like growth factor-I and insulin-like growth factor binding protein than was induced by daily injections of the same amount of rhGH in solution. After three monthly injections of one of the formulations, both the rhGH and IGF-I levels remained elevated for nearly 90 days. Immunogenicity of the rhGH released from this formulation, as assessed by the incidence of seroconversion to hGH and the titer of anti-hGH antibody in both the rhesus monkeys and transgenic mice expressing rhGH, was no greater than that of the unencapsulated protein. In addition, the microsphere injection sites appeared normal by macroscopic evaluation between 1 to 2 mo after microsphere administration and by microscopic evaluation between 2 to 3 mo. These results show that serum levels of a therapeutic protein can be sustained for an extended period when encapsulated into different formulations of injectable, biodegradable microspheres.     

Effects of supervisor and supervisee theoretical orientation and supervisor-supervisee matching on interns' perceptions of supervision.

Effects of supervisor and supervisee theoretical orientation on supervisees' perceptions of supervisors' models, roles, and foci were studied, as were variables influencing quality of supervision and supervisee autonomy. 84 interns from 32 nationwide training sites were surveyed. Cognitive-behavioral supervisors were perceived to be in a consultant role and to focus on skills and strategies more than were humanistic, psychodynamic, and existential supervisors, who were perceived more as using the relationship model, playing the therapist role, and focusing on conceptualization. Supervisors were not perceived to differ in their use of growth and skill development models, teacher role, and focus on the supervisee. Women were perceived as more effective supervisors than were men. Perceived effectiveness was predicted by theoretical match and similarity. Supervisee autonomy was predicted by theoretical similarity, low supervisor adherence to theory, and unmatched gender. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Differential DNA Methylation in Prostate Tumors from Puerto Rican Men

In 2020, approximately 191,930 new prostate cancer (PCa) cases are estimated in the United States (US). Hispanic/Latinos (H/L) are the second largest racial/ethnic group in the US. This study aims to assess methylation patterns between aggressive and indolent PCa including DNA repair genes along with ancestry proportions. Prostate tumors classified as aggressive (n = 11) and indolent (n = 13) on the basis of the Gleason score were collected. Tumor and adjacent normal tissue were annotated on H&E (Haemotoxylin and Eosin) slides and extracted by macro-dissection. Methylation patterns were assessed using the Illumina 850K DNA methylation platform. Raw data were processed using the Bioconductor package. Global ancestry proportions were estimated using ADMIXTURE (k = 3). One hundred eight genes including AOX1 were differentially methylated in tumor samples. Regarding the PCa aggressiveness, six hypermethylated genes (RREB1, FAM71F2, JMJD1C, COL5A3, RAE1, and GABRQ) and 11 hypomethylated genes (COL9A2, FAM179A, SLC17A2, PDE10A, PLEKHS1, TNNI2, OR51A4, RNF169, SPNS2, ADAMTSL5, and CYP4F12) were identified. Two significant differentially methylated DNA repair genes, JMJD1C and RNF169, were found. Ancestry proportion results for African, European, and Indigenous American were 24.1%, 64.2%, and 11.7%, respectively. The identification of DNA methylation patterns related to PCa in H/L men along with specific patterns related to aggressiveness and DNA repair constitutes a pivotal effort for the understanding of PCa in this population.     

Sulfhydryl reagents and cAMP-dependent kinase increase the sensitivity of the inositol 1,4,5-trisphosphate receptor in hepatocytes

Sulfhydryl reagents such as tert-butyl hydroperoxide (TBHP) have been shown to increase cytosolic Ca2+ concentration ([Ca2+]i) in rat hepatocytes in a way that resembles responses to Ca(2+)-mobilizing hormones (Saikada, I., Thomas, A. P., and Farber, J. L. (1991) J. Biol. Chem. 266, 717-722; Rooney, T. A., Renard, D. C., Sass, E. J., and Thomas, A. P. (1991) J. Biol. Chem. 266, 12272-12282) and to increase the amount of Ca2+ released by inositol 1,4,5-trisphosphate ((1,4,5)IP3) from permeable rat liver cells (Rooney et al., 1991, op. cit.; Missiaen, L., Taylor, C. W., and Berridge, M. J. (1991) Nature 352, 241-244; Renard, D. C., Seitz, M. B., and Thomas, A. P. (1992) Biochem. J. 284, 507-512). The effects of sulfhydryl reagents were studied in fura-2-injected rat and guinea pig hepatocytes and compared with the actions of cAMP (Burgess, G. M., Bird, G. St. J., Obie, J. F., and Putney, J. W., Jr. (1991) J. Biol. Chem. 261, 4772-4781). In rat liver cells, the increases in [Ca2+]i induced by TBHP and thimerosal were prevented by microinjection of the cells with the (1,4,5)IP3 receptor antagonist heparin. In guinea pig hepatocytes, TBHP was not able to increase [Ca2+]i unless the cells were pretreated with angiotensin II to raise endogenous levels of (1,4,5)IP3 or were first injected with a sub-threshold concentration of inositol 2,4,5-trisphosphate ((2,4,5)IP3). The responses to TBHP in (2,4,5)IP3-injected guinea pig cells were also blocked by heparin. In many respects, the actions of TBHP appeared to be similar to those of cAMP, which has previously been shown to increase sensitivity to (1,4,5)IP3 in intact guinea pig hepatocytes (Burgess et al., 1991, op. cit.). TBHP also mimicked the effect of cAMP-dependent kinase (PKA) in permeabilized guinea pig hepatocytes by increasing the amount of Ca2+ released by (1,4,5)IP3. The responses to TBHP and cAMP in (2,4,5)IP3-injected guinea pig hepatocytes differed, however, in that the increase in [Ca2+]i evoked by elevating intracellular cAMP was greatly reduced by Wiptide, an inhibitor of PKA, while Wiptide had no effect on the Ca2+ transients induced by TBHP. This provides evidence that the sensitizing effect of TBHP is not mediated by PKA and is more likely to be a direct effect on the inositol trisphosphate receptor. It is possible, however, that the sulfhydryl reagents and PKA act on a common regulatory site on the receptor protein.     

Real-Time Spectrum Analysis and Display of Directional Doppler Ultrasound Blood Velocity Signals

Development of a real-time system for three-dimensional display of Doppler shifted frequencies within an ultrasound flowmeter signal is described. The device is based on a fast Fourier transform (FFT) spectrum analyzer which provides both high frequency and time resolution over a variable range of frequencies up to 40 kHz. Signal preprocessing filters unwanted low frequency artifacts due to arterial wail motion and automatically controls input gain to limit peak amplitudes while matched phase shift networks translate forward and reverse components for a directional output. A digital first moment is calculated and the display may be viewed in a continuous or freeze frame mode. Clinical applications underway have shown improved detail and accuracy in the use of ultrasound for diagnosing various vascular abnormalities.     

A Multimodal Exploration of Engineering Students' Emotions and Electrodermal Activity in Design Activities

Background

This exploratory study uses multimodal approaches to explore undergraduate student engagement via topic emotions and electrodermal activity (EDA) in different engineering design method activities and with different instructional delivery formats (e.g., lecture vs. active learning).

Purpose/Hypothesis

The goal of this research is to improve our understanding of how students respond, via engagement, to their engineering design activities during class. This study hypothesizes that students would experience no self‐reported mean changes in topic emotions from their preassessment scores for each engineering design topic and instructional format nor would electrodermal activities (EDA) associate to these topic emotions throughout the design activities.

Design/Method

Eighty‐eight freshmen engineering students completed online pretopic and posttopic emotions surveys for five engineering design activities. A subset of 14–18 participants, the focal point of this study, wore an EDA sensor while completing the surveys and participating in these sessions.

Results

Preliminary findings suggest that EDA increased for individual and collaborative active learning activities compared to lectures. No significant changes in EDA were found between individual and collaborative active learning activities. Moderate negative correlations were found between EDA and negative topic emotions in the first engineering design activity but not across the rest. At the end of the semester, active learning activities showed higher effect sizes indicating a re‐enforcement of students' engagement in the engineering design method activities.

Conclusion

This study provides initial results showing how multimodal approaches can help researchers understand students' closer‐to‐real‐time engagement in engineering design topics and instructional delivery formats.