Relocation of a Ca2+-dependent protein kinase activity during pollen tube reorientation

Pollen tube reorientation is a dynamic cellular event that is crucial for successful fertilization. We have shown previously that pollen tube orientation is regulated by cytosolic free calcium ([Ca2+]c). In this paper, we studied the activity of a Ca2+-dependent protein kinase during reorientation. The kinase activity was assayed in living cells by using confocal ratio imaging of BODIPY FL bisindolylmaleimide. We found that growing pollen tubes exhibited higher protein kinase activity in the apical region, whereas nongrowing cells showed uniform distribution. Modification of growth direction by diffusion of inhibitors/activators from a micropipette showed the spatial redistribution of kinase activity to predict the new growth orientation. Localized increases in [Ca2+]c induced by photolysis of caged Ca2+ that led to reorientation also increased kinase activity. Molecular and immunological assays suggest that this kinase may show some functional homology with protein kinase C. We suggest that the tip-localized gradient of kinase activity promotes Ca2+-mediated exocytosis and may act to regulate Ca2+ channel activity.     

Interdependent MHC-DRB exon-plus-intron evolution in artiodactyls

Exon 2 sequences of an expressed MHC-DRB locus from sheep were examined for polymorphisms in both the antigen-binding regions and the adjacent intronic mixed simple tandem repeat. Twenty-one novel exon 2 Ovar-DRB alleles were identified. Short nucleotide motifs are extensively shared between certain exon 2 regions of Ovar-DRB alleles. The simple repeat variations, the number of different amino acids at usually polymorphic sites, and the number of silent substitutions were reduced in the intraspecies analyses of sheep DRB sequences, compared with those of cattle and goats. It was paradoxical that the abundance of different sheep alleles was similar to that of cattle and goats. This paradox may be explained by postulating a relatively small number of "ancient" alleles, with the present-day Ovar-DRB alleles being generated by reciprocal exchange of nucleotide motifs. At the antigen-binding sites, new combinations of amino acids were maintained in Ovar-DRB alleles by strong positive selection. In sheep--and less pronounced in goats and cattle--the DRB alleles can be divided into two groups. In one group, silent substitutions are increased when compared with the other. This suggests separate evolutionary pathways for certain groups of DRB alleles within a species. The simple repetitive sequences are also discussed with respect to the evolution of DRB alleles.      

The effect of selective phosphodiesterase inhibitors,alone and in combination,on a murine model of allergic asthma

BackgroundThe anti-inflammatory effects of the selective phosphodiesterase (PDE) inhibitors cilostazol (PDE 3), RO 20-1724 (PDE 4) and sildenafil (PDE 5) were examined in a murine model of allergic asthma. These compounds were used alone and in combination to determine any potential synergism, with dexamethasone included as a positive control.MethodsControl and ovalbumin sensitised Balb/C mice were administered orally with each of the possible combinations of drugs at a dose of 3 mg/Kg for 10 days.ResultsWhen used alone, RO 20-1724 significantly reduced eosinophil influx into lungs and lowered tumour necrosis factor-α, interleukin-4 and interleukin-5 levels in the bronchoalveolar lavage fluid when compared to untreated mice. Treatment with cilostazol or sildenafil did not significantly inhibit any markers of inflammation measured. Combining any of these PDE inhibitors produced no additive or synergistic effects. Indeed, the anti-inflammatory effects of RO 20-1724 were attenuated by co-administration of either cilostazol or sildenafil.ConclusionsThese results suggest that concurrent treatment with a PDE 3 and/or PDE 5 inhibitor will reduce the anti-inflammatory effectiveness of a PDE 4 inhibitor.     

T-cell activation without proliferation in juvenile idiopathic arthritis

A study was done to determine if the differentiation and activation phenotype of T cells in synovial fluid (SF) from patients with juvenile idiopathic arthritis (JIA) is associated with T-cell proliferation in situ. Mononuclear cells were isolated from 44 paired samples of peripheral blood and SF. Differentiation and activation markers were determined on CD4 and CD8 T cells by flow cytometry. Cell-cycle analysis was performed by propidium iodide staining, and surface-marker expression was also assessed after culture of the T cells under conditions similar to those found in the synovial compartment. The majority of the T cells in the SF were CD45RO⁺CD45RB^dull. There was greater expression of the activation markers CD69, HLA-DR, CD25 and CD71 on T cells from SF than on those from peripheral blood. Actively dividing cells accounted for less than 1% of the total T-cell population in SF. The presence or absence of IL-16 in T-cell cultures with SF or in a hypoxic environment did not affect the expression of markers of T-cell activation. T cells from the SF of patients with JIA were highly differentiated and expressed early and late markers of activation with little evidence of in situ proliferation. This observation refines and extends previous reports of the SF T-cell phenotype in JIA and may have important implications for our understanding of chronic inflammation.     

Cochlear function in mice following inhalation of brevetoxin-3

Brevetoxin-3 was shown previously to adversely affect central auditory function in goldfish. The present study evaluated the effects of exposure to this agent on cochlear function in mice using the 2f₁-f₂ distortion-product otoacoustic emission (DPOAE). Towards this end, inbred CBA/CaJ mice were exposed to a relatively high concentration of brevetoxin-3 (~400 g/m³) by nose-only inhalation for a 2-h period. Further, a subset of these mice received a second exposure a day later that lasted for an additional 4 h. Mice exposed only once for 2 h did not exhibit any notable cochlear effects. Similarly, mice exposed two times, for a cumulative dose of 6 h, exhibited essentially no change in DPOAE levels.     

Deciphering membrane protein structures from protein sequences

ABSTRACT: Co-evolving positions within protein sequences have been used as spatial constraints to develop a computational approach for modeling membrane protein structures.     

Automated determination of bone age and bone mineral density in patients with juvenile idiopathic arthritis: a feasibility study

Introduction

Chronic inflammation combined with glucocorticoid treatment and immobilization puts juvenile idiopathic arthritis (JIA) patients at risk of impaired growth and reduced bone mineral density (BMD). Conventional methods for evaluating bone age and BMD are time-consuming or come with additional costs and radiation exposure. In addition, an automated measurement of bone age and BMD is likely to be more consistent than visual evaluation. In this study, we aimed to evaluate the feasibility of an automated method for determination of bone age and (cortical) bone mineral density (cBMD) in severely affected JIA patients. A secondary objective was to describe bone age and cBMD in this specific JIA population eligible for biologic treatment.

Methods

In total, 69 patients with standard hand radiographs at the start of etanercept treatment and of calendar age within the reliability ranges (2.5 to 17 years for boys and 2 to 15 years for girls) were extracted from the Dutch Arthritis and Biologicals in Children register. Radiographs were analyzed using the BoneXpert method, thus automatically determining bone age and cBMD expressed as bone health index (BHI). Agreement between measurements of the left- and right-hand radiographs and a repeated measurement of the left hand were assessed with the intraclass correlation coefficient (ICC). Regression analysis was used to identify variables associated with Z-scores of bone age and BHI.

Results

The BoneXpert method was reliable in the evaluation of radiographs of 67 patients (radiographs of 2 patients were rejected because of poor image quality). Agreement between left- and right-hand radiographs (ICC = 0.838 to 0.996) and repeated measurements (ICC = 0.999 to 1.000) was good. Mean Z-scores of bone age (−0.36, P = 0.051) and BHI (−0.85, P < 0.001) were lower compared to the healthy population. Glucocorticoid use was associated with delayed bone age (0.79 standard deviation (SD), P = 0.028), and male gender was associated with a lower Z-score of BHI (0.65 SD, P = 0.021).

Conclusions

BoneXpert is an easy-to-use method for assessing bone age and cBMD in patients with JIA, provided that radiographs are of reasonable quality and patients’ bone age lies within the age ranges of the program. The population investigated had delayed bone maturation and lower cBMD than healthy children.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-014-0424-1) contains supplementary material, which is available to authorized users.