Stimulation of Cultured Melanocytes in Medium Containing a Serum Substitute: Ultroser-G

Melanocyte cultures were established and maintained routinely in Ham's F-10 medium containing 12-O-tetradecanoyl-phorbol-13-acetate (TPA), isobutylmethylxanthine (IBMX), cholera toxin (CT) and fetal calf serum (FCS). Three serum substitutes (Ultroser-G, Nutridoma-Hu and Nutricyte-H) were tested in order to obtain a medium without FCS having a more constant composition. Melanocyte proliferation was examined in long-term culture experiments by in situ cell counts at different periods of time. Only with Ultroser-G (1-2%) was the proliferation of melanocytes maintained without both FCS and CT, whereas the addition of the other two serum substitutes resulted in stabilization of melanocyte densities in the cultures up to 28 days. In the medium containing 1% Ultroser-G and IBMX without TPA minimal or no increases in melanocyte density were found. Addition of basic fibroblast growth factor (bFGF, 1 ng/ml) to the medium without TPA resulted in a partial restimulation of growth in different experiments. In this system with 1% Ultroser-G and 1 ng/ml bFGF, IBMX could also be replaced by other factors (dbcAMP LTC4 and a purified form of α-melanocyte stimulating hormone). The culture medium with 1% Ultroser-G containing TPA and IBMX is now used for routine melanocyte culture. In this medium TPN/IBMX can easily be replaced by bFGF/dbcAMP with optimal growth stimulation. The combination bFGF/α-MSH and other more physiological stimulators offers an alternative to study responses of melanocytes in culture with respect to proliferation, metabolism, and phenotype.     

Oxygen cost of inspiratory loading: resistive vs. elastic

We measured the O2 cost of breathing (VO2resp) against external inspiratory elastic (E) and resistive loads (R) when end-expiratory lung volume, tidal volume, breathing frequency, work rate, and pressure-time product were matched in each of six pairs of runs in six subjects. During E, peak inspiratory mouth pressure was 65.7 +/- 1.8% (SD) of the maximum at functional residual capacity. However, during resistive runs, peak inspiratory mouth pressure was 41.1 +/- 2.8% of the maximum at functional residual capacity. In 36 paired runs, where both work rate and pressure-time product were within 10%, VO2resp for E was less than for R (81 and 96 ml/min, respectively; P less than 0.01). During loaded and unloaded breathing with the same tidal volume, we measured the changes in anteroposterior diameter of the lower rib cage in five subjects. In four subjects we also recorded the electromyograms of several fixator and stabilizing muscles. During E and R, the change in anteroposterior diameter of the lower rib cage was -116 +/- 5 and -45 +/- 4% (SE), respectively, of the unloaded value (P less than 0.01), indicating greater deformation during E. Although the peak electromyographic activity was 72 +/- 16% greater during E (P less than 0.01), there was no difference between the loads for area under the electromyogram time curve (P greater than 0.05). However, the time to 50% peak activity was less during R (P less than 0.02). We conclude that, even when work rate and pressure-time product are matched, VO2resp during R is greater than that during E. This difference may be due to preferential recruitment of faster and less efficient muscle fibers.     

Intralumenal sarcoplasmic reticulum Ca(2+)-binding proteins

The sarcoplasmic reticulum (SR) controls the level of intracellular Ca2+ in cardiac and skeletal muscle by storing and releasing Ca2+. A set of intralumenal SR Ca(2+)-binding proteins has been identified that may serve important roles in SR Ca2+ storage and mobilization. The most prominent of these SR proteins, calsequestrin, is discretely localized to junctional SR. Other intralumenal proteins are more widely distributed throughout the SR. All of these intralumenal SR Ca(2+)-binding proteins are acidic, stain blue with dye Stains-All, and appear to be substrates for casein kinase II. The biochemistry and cell biology of lumenal SR proteins may conform to a paradigm now emerging from the study of endoplasmic reticulum proteins.     

Determinants of exercise performance in normal men with externally imposed expiratory flow limitation.

To understand how externally applied expiratory flow limitation (EFL) leads to impaired exercise performance and dyspnea, we studied six healthy males during control incremental exercise to exhaustion (C) and with EFL at approximately 1. We measured volume at the mouth (Vm), esophageal, gastric and transdiaphragmatic (Pdi) pressures, maximal exercise power (W(max)) and the difference (Delta) in Borg scale ratings of breathlessness between C and EFL exercise. Optoelectronic plethysmography measured chest wall and lung volume (VL). From Campbell diagrams, we measured alveolar (PA) and expiratory muscle (Pmus) pressures, and from Pdi and abdominal motion, an index of diaphragmatic power (W(di)). Four subjects hyperinflated and two did not. EFL limited performance equally to 65% W(max) with Borg = 9-10 in both. At EFL W(max), inspiratory time (TI) was 0.66s +/- 0.08, expiratory time (TE) 2.12 +/- 0.26 s, Pmus approximately 40 cmH2O and DeltaVL-DeltaVm = 488.7 +/- 74.1 ml. From PA and VL, we calculated compressed gas volume (VC) = 163.0 +/- 4.6 ml. The difference, DeltaVL-DeltaVm-VC (estimated blood volume shift) was 326 ml +/- 66 or 7.2 ml/cmH2O PA. The high Pmus and long TE mimicked a Valsalva maneuver from which the short TI did not allow recovery. Multiple stepwise linear regression revealed that the difference between C and EFL Pmus accounted for 70.3% of the variance in DeltaBorg. DeltaW(di) added 12.5%. We conclude that high expiratory pressures cause severe dyspnea and the possibility of adverse circulatory events, both of which would impair exercise performance.     

Mass spectrometry of cardiac calsequestrin characterizes microheterogeneity unique to heart and indicative of complex intracellular transit

Cardiac calsequestrin concentrates in junctional sarcoplasmic reticulum in heart and skeletal muscle cells by an undefined mechanism. During transit through the secretory pathway, it undergoes an as yet uncharacterized glycosylation and acquires phosphate on CK2-sensitive sites. In this study, we have shown that active calsequestrin phosphorylation occurred in nonmuscle cells as well as muscle cells, reflecting a widespread cellular process. To characterize this post-translational modification and resolve individual molecular mass species, we subjected purified calsequestrin to mass spectrometry using electrospray ionization. Mass spectra showed that calsequestrin glycan structure in nonmuscle cells was that expected for an endoplasmic reticulum-localized glycoprotein and showed that each glycoform existed as four mass peaks representing molecules that also had 0-3 phosphorylation sites occupied. In heart, mass peaks indicated carbohydrate modifications characteristic of transit through Golgi compartments. Phosphorylation did not occur on every glycoform present, suggesting a far more complex movement of calsequestrin molecules in heart cells. Significant amounts of calsequestrin contained glycan with only a single mannose residue, indicative of a novel post-endoplasmic reticulum mannosidase activity. In conclusion, glyco- and phosphoforms of calsequestrin chart a complex cellular transport in heart, with calsequestrin following trafficking pathways not present or not accessible to the same molecules in nonmuscle.     

GRP94 hyperglycosylation and phosphorylation in Sf21 cells

GRP94 is an inducible resident endoplasmic reticulum/sarcoplasmic reticulum (ER/SR) glycoprotein that functions as a protein chaperone and Ca(2+) regulator. GRP94 has been reported to be a substrate for protein kinase CK2 in vitro, although its phosphorylation in intact cells remains unreported. In Sf21 insect cells, overexpression of canine GRP94 led to the appearance of a multiplet of three or more molecular-mass isoforms which was reduced to a single mobility form following treatment of cells with tunicamycin, suggesting stable accumulations of consecutively modified protein. Metabolic labeling of Sf21 cells with (32)P(i) led to a constitutive phosphorylation of GRP94 which, based upon phosphopeptide mapping, occurred specifically on CK2-sensitive sites. Among the GRP94 multiplet, however, only the lowest mobility form of GRP94 was phosphorylated, even though in vitro phosphorylation of GRP94 by CK2 led to phosphorylation of all glycosylated forms. The (32)P(i) incorporation into GRP94 indicated a slow turnover of phosphate incorporation that was unaffected by inhibition of biosynthesis, resulting in a steady-state level of phospho-GRP94 on CK2 sites. These data support a role for protein kinase CK2 in the cell biology for GRP94 and other resident ER/SR proteins that may occur in ER compartments.     

OPTIMAS-DW: A comprehensive transcriptomics,metabolomics, ionomics,proteomics and phenomics data resource for maize

Background

The wild barley Hordeum chilense fulfills some requirements for being a useful tool to investigate the endosperm yellow pigment content (YPC) in the Triticeae including its diploid constitution, the availability of genetic resources (addition and deletion stocks and a high density genetic map) and, especially, its high seed YPC not silenced in tritordeums (amphiploids derived from H. chilense and wheat). Thus, the aim of this work was to test the utility of the H. chilense genome for investigating the YPC in the Triticeae.

Results

Twelve genes related to endosperm carotenoid content and/or YPC in grasses (Dxr, Hdr [synonym ispH], Ggpps1, Psy2, Psy3, Pds, Zds, e-Lcy, b-Lcy, Hyd3, Ccd1 and Ppo1) were identified, and mapped in H. chilense using rice genes to identify orthologs from barley, wheat, sorghum and maize. Macrocolinearity studies revealed that gene positions were in agreement in H. vulgare and H. chilense. Additionally, three main regions associated with YPC were identified in chromosomes 2H^ch, 3H^ch and 7H^ch in H. chilense, the former being the most significant one.

Conclusions

The results obtained are consistent with previous findings in wheat and suggest that Ggpps1, Zds and Hyd3 on chromosome 2H^ch may be considered candidate genes in wheat for further studies in YPC improvement. Considering the syntenic location of carotenoid genes in H. chilense, we have concluded that the H^ch genome may constitute a valuable tool for YPC studies in the Triticeae.

     

Moving instead of asking? Performance-based tests and BASFI-questionnaire measure different aspects of physical function in ankylosing spondylitis

Introduction

Ankylosing Spondylitis (AS) is characterised by limitations in physical function. The Bath Ankylosing Spondylitis Functional Index (BASFI) is considered to be the gold-standard to assess physical function in AS patients. However, the BASFI questionnaire is a self-reported outcome measure and susceptible to subjective interpretation (under- or over-estimation). More objective outcome measures, like performance-based tests, could provide an objective outcome measurement for the evaluation of limitations in physical function. Therefore, the primary aim of this study was to determine the association between performance-based measures and the BASFI questionnaire.

Methods

In this cross-sectional study 126 AS patients completed the BASFI questionnaire and eight performance-based tests based on BASFI-items. Each test received three scores: one for performance (time or points) and a score for exertion and pain experienced during performance (using modified Borg-scale and VAS 0-100 mm, respectively). Linear regression analyses were used to assess the associations between the BASFI questionnaire and performance-based tests.

Results

The univariable association between performance and BASFI-score was moderate with a R-square of 0.31 and Beta of 0.56 (p's < 0.05). In a multivariable analysis, the association between performance, exertion and pain on the one hand and BASFI-score on the other was assessed; R-square increased to 0.54: the Beta's for exertion and pain during performance were 0.38 and 0.26, respectively; the Beta for performance decreased to 0.19 (p's < 0.05).

Conclusions

This study demonstrates that alongside actual performance, patients seem to incorporate exertion and pain in their assessment of perceived physical function on the BASFI questionnaire. Performance-based tests could provide an objective outcome measurement for the evaluation of physical function and give relevant new information in addition to the BASFI questionnaire.     

Designing a strategy to implement cost-effective blood transfusion management in elective hip and knee arthroplasties: A study protocol

ABSTRACT: BACKGROUND: Total hip and knee arthroplasties are two of the most commonly performed procedures in orthopedic surgery. Different blood-saving measures (BSMs) are used to reduce the often-needed allogenic blood transfusions in these procedures. A recent large randomized controlled trial showed it is not cost effective to use the BSMs of erythropoietin and perioperative autologous blood salvage in elective primary hip and knee arthroplasties. Despite dissemination of these study results, medical professionals keep using these BSMs. To actually change practice, an implementation strategy is needed that is based on a good understanding of target groups and settings and the psychological constructs that predict behavior of medical professionals. However, detailed insight into these issuses is lacking. Therefore, this study aims to explore which groups of professionals should be targeted at which settings, as well as relevant barriers and facilitators that should be taken into acount in the strategy to implement evidence-based, cost-effective blood transfusion management and to de-implement BSMs. METHODS: The study consists of three phases. First, a questionnaire survey among all Dutch orthopedic hospital departments and independent treatment centers (n?=?99) will be conducted to analyze current blood management practice. Second, semistructured interviews will be held among 10 orthopedic surgeons and 10 anesthesiologists to identify barriers and facilitators that are relevant for the uptake of cost-effective blood transfusion management. Interview questions will be based on the Theoretical Domains Interview framework. The interviews will be followed by a questionnaire survey among 800 medical professionals in orthopedics and anesthesiology (400 professionals per discipline) in which the identified barriers and facilitators will be ranked by frequency and importance. Finally, an implementation strategy will be developed based on the results from the previous phases, using principles of intervention mapping and an expert panel. DISCUSSION: The developed strategy for cost-effective blood transfusion management by de-implementing BSMs is likely to reduce costs for elective hip and knee arthroplasties. In addition, this study will lead to generalized knowledge regarding relevant factors for the de-implementation of non-cost-effective interventions and insight in the differences between implementation and de-implementation strategies.