Different role of intracellular loops of glucagon-like peptide-1 receptor in G-protein coupling

Previous studies revealed the importance of the third intracellular loop of glucagon-like peptide-1 receptor (GLP-1R) in coupling to G(s) and G(i1) proteins. In order to further study the signaling mechanisms of GLP-1R, we tested three peptides, corresponding to the sequences of the first (IC(1)), the second (IC(2)), and the third (IC(3)) intracellular loop of GLP-1R, for their interactions with heterotrimeric G-proteins of different types (G(alphas), G(alphao), G(alphai1), and G(alpha11) plus G(beta1gamma2)) overexpressed in sf9 cells. IC(3) peptide powerfully stimulates all types of tested G-proteins, whereas IC(1) and IC(2) peptides show differential effects on G-proteins. Both IC(1) and IC(2) peptides activate G(s) and cooperate with IC(3) peptide in its stimulation. G(o) is not affected by IC(1) and IC(2). G(i1) and G(11) are not affected by IC(1), but are activated by IC(2), which in activation cooperates with IC(3). We suggest that GLP-1R is not coupled only to G(s) and G(i1), as shown previously, but also to G(o) and G(11). IC(3) loop is the main switch that mediates signaling via GLP-1R to G-proteins, while IC(1) and IC(2) loops are important in discrimination between different types of G-proteins.     

Phytochelatin synthase catalyzes key step in turnover of glutathione conjugates

Conjugation of xenobiotic compounds and endogenous metabolites to glutathione is an ubiquitous process in eukaryotes. In animals, the first and rate-limiting step of glutathione-S-conjugate metabolism is characterized by the removal of the aminoterminal glutamic acid residue of glutathione. In plants, however, glutathione-S-conjugates are generally metabolized by removal of the carboxylterminal glycine residue of the tripeptide glutathione to give rise to the S-glutamylcysteinyl-derivative. Purification of the glutathione-conjugate catabolizing activity from cell suspension cultures of the plant Silene cucubalus indicated that phytochelatin synthase catalyzes the first step of the pathway. Heterologously expressed phytochelatin synthase from Arabidopsis efficiently converted S-bima ne-glutathione to S-bimane-glutamylcysteine, the formation of which was unequivocally identified by mass spectrometry. No further products, such as S-derivatives of phytochelatins, were observed. Several different glutathione-S-conjugates served as substrates for the enzyme and were processed to the corresponding glutamylcysteinyl-adducts. Affinity-purified phytochelatin synthase preparations required divalent heavy metal ions such as Cd(2+), Zn(2+) or Cu(2+) for detectable turnover of glutathione-S-conjugates. Characterization of the enzymatic properties of phytochelatin synthase argues for both cellular functions of the gamma-glutamylcysteinyl-dipeptidyltransferase: (1) formation of heavy-metal binding peptides and (2) degradation of glutathione-S-conjugates. Mechanistically, the former role is the result of gamma-glutamylcysteinyl transpeptidation onto glutathione or derivatives thereof, while the catabolic function reflects transpeptidation of S-glutamylcysteinyl-adducts onto the acceptor molecule water. Thus, phytochelatin synthase seems to fulfil a second crucial role in glutathione metabolism.     

The outcome of autologous chondrocyte transplantation treatment of cartilage lesions in the knee

Recent results of the clinical outcome of autologous chondrocyte transplantation (ACT) treatment in a group of 28 patients with focal femoral condyle cartilage lesions revealed a correlation trend with the quality of the in vitro cell culture matrix-protein synthesis. No impact of the patients' age and chondrocyte cryopreservation prior to implantation was observed. Further studies are needed to confirm the preliminary results.     

Sensitivity and flexibility of regular and chaotic calcium oscillations

Sensitivity and flexibility are important properties of biological systems. These properties are here investigated for intracellular calcium oscillations. For a particular model, we comparatively investigate sensitivity and flexibility of regular and chaotic Ca(2+) oscillations. For this model, we obtain two main results. First, sensitivity of the model system to parameter shifting does not depend on the complexity of Ca(2+) oscillations. We observe, however, that both regular and chaotic Ca(2+) oscillations are highly sensitive in regions close to bifurcation points. Second, also flexibility of Ca(2+) oscillations does not significantly depend on the type of Ca(2+) oscillations. Our results show that regular as well as chaotic Ca(2+) oscillations in the studied model are highly flexible in regimes with weak dissipation. Both results are discussed in the sense of possible biological importance.     

Exocytotic release of ATP from cultured astrocytes

Astrocytes appear to communicate with each other as well as with neurons via ATP. However, the mechanisms of ATP release are controversial. To explore whether stimuli that increase [Ca(2+)](i) also trigger vesicular ATP release from astrocytes, we labeled ATP-containing vesicles with the fluorescent dye quinacrine, which exhibited a significant co-localization with atrial natriuretic peptide. The confocal microscopy study revealed that quinacrine-loaded vesicles displayed mainly non-directional spontaneous mobility with relatively short track lengths and small maximal displacements, whereas 4% of vesicles exhibited directional mobility. After ionomycin stimulation only non-directional vesicle mobility could be observed, indicating that an increase in [Ca(2+)](i) attenuated vesicle mobility. Total internal reflection fluorescence (TIRF) imaging in combination with epifluorescence showed that a high percentage of fluorescently labeled vesicles underwent fusion with the plasma membrane after stimulation with glutamate or ionomycin and that this event was Ca(2+)-dependent. This was confirmed by patch-clamp studies on HEK-293T cells transfected with P2X(3) receptor, used as sniffers for ATP release from astrocytes. Glutamate stimulation of astrocytes was followed by an increase in the incidence of small transient inward currents in sniffers, reminiscent of postsynaptic quantal events observed at synapses. Their incidence was highly dependent on extracellular Ca(2+). Collectively, these findings indicate that glutamate-stimulated ATP release from astrocytes was most likely exocytotic and that after stimulation the fraction of quinacrine-loaded vesicles, spontaneously exhibiting directional mobility, disappeared.     

Establishing the stochastic nature of intracellular calcium oscillations from experimental data

Calcium has been established as a key messenger in both intra- and intercellular signaling. Experimentally observed intracellular calcium responses to different agonists show a variety of behaviors from simple spiking to complex oscillatory regimes. Here we study typical experimental traces of calcium oscillations in hepatocytes obtained in response to phenylephrine and ATP. The traces were analyzed with methods of nonlinear time series analysis in order to determine the stochastic/deterministic nature of the intracellular calcium oscillations. Despite the fact that the oscillations appear, visually, to be deterministic yet perturbed by noise, our analyses provide strong evidence that the measured calcium traces in hepatocytes are prevalently of stochastic nature. In particular, bursting calcium oscillations are temporally correlated Gaussian series distorted by a monotonic, instantaneous, time-independent function, whilst the spiking behavior appears to have a dynamical nonlinear component whereby the overall determinism level is still low. The biological importance of this finding is discussed in relation to the mechanisms incorporated in mathematical models as well as the role of stochasticity and determinism at cellular and tissue levels which resemble typical statistical and thermodynamic effects in physics.     

Urgent Ultrasound Guided Hemodynamic Assessments by a Pediatric Medical Emergency Team: A Pilot Study

Purpose

To determine the feasibility of using the Ultrasound Cardiac Output Monitor (USCOM) as an adjunct during hemodynamic assessments by a pediatric medical emergency team (PMET).

Methods

Pediatric in-patients at McMaster Children’s Hospital aged under 18 years requiring urgent PMET consultation, were eligible. Patients with known cardiac outflow valve defects, Pediatric Critical Care Unit in-patients, and those in cardiorespiratory arrest, were excluded. The primary outcome was feasibility, and the ease of USCOM transport and application as assessed by a self-administered user questionnaire. Secondary outcomes included the quality of USCOM measurements, and agreement in clinical versus USCOM-derived assessments.

Results

Forty-one patients from 85 eligible PMET consultations were enrolled between March and August 2011. A total of 55 USCOM assessments were performed on 36 of 41 (87.8%) participants. USCOM could not be completed in 5 (12.2%) participants due to patient agitation (n = 4) and emergent care (n = 1). USCOM was reported as easy to transport and apply by 97.4% and 94.7% of respondents respectively, not obstructive to patient care by 94.7%, and yielded timely measurements by 84.2% respondents. USCOM tracings were of good quality in 41 (75.9%) assessments. Agreement between clinical and USCOM-derived hemodynamic assessments by two independent raters was poor (Rater 1: κ = 0.094; Rater 2: κ = 0.146).

Conclusion

USCOM can be applied by a PMET during urgent hemodynamic assessments in children. While USCOM has been validated in stable children, its role in guiding hemodynamic resuscitation and informing therapeutic goals in a hemodynamically unstable pediatric population requires further investigation.     

ERBB4 Promoter Polymorphism Is Associated with Poor Distant Disease-Free Survival in High-Risk Early Breast Cancer

A number of genetic variants have been linked to increased risk of breast cancer. Little is, however, known about the prognostic significance of hereditary factors. Here, we investigated the frequency and prognostic significance of two ERBB4 promoter region variants, −782G>T (rs62626348) and −815A>T (rs62626347), in a cohort of 1010 breast cancer patients. The frequency of nine previously described somatic ERBB4 kinase domain mutations was also analyzed. Clinical material used in the study consisted of samples from the phase III, adjuvant, FinHer breast cancer trial involving 1010 women. Tumor DNA samples were genotyped for ERBB4 variants and somatic mutations using matrix-assisted laser desorption ionization/time of flight mass spectrometry. Paraffin-embedded tumor sections from all patients were immunohistochemically stained for ErbB4 expression. Association of ERBB4 genotype to distant disease-free survival (DDFS) was assessed using Kaplan-Meier and Cox regression analyses. Genotyping was successful for 91–93% of the 1010 samples. Frequencies observed for the ERBB4 variants were 2.5% and 1.3% for −782G>T and −815A>T, respectively. Variant −815A>T was significantly associated with poor survival (HR  = 2.86 [95% CI 1.15–6.67], P = 0.017). In contrast, variant −782G>T was associated with well-differentiated cancer (P = 0.019). Two (0.2%) ERBB4 kinase domain mutations were found, both of which have previously been shown to be functional and promote cancer cell growth in vitro. These data present the germ-line ERBB4 variant −815A>T as a novel prognostic marker in high-risk early breast cancer and indicate the presence of rare but potentially oncogenic somatic ERBB4 mutations in breast cancer.