Expression of the full-length rabbit prolactin receptor and its specific domains in baculovirus infected insect cells

The prolactin receptor is a membrane protein mainly involved in the development of the mammary gland and in lactation in mammals. We used specific cDNA constructs and the insect/baculovirus expression system and produced independently and in large amounts several recombinant forms of the rabbit mammary gland prolactin receptor: the full-length receptor (L1, L2), a truncated membrane form (S), a secretable form of the extracellular domain (E) and two forms of the intracellular domain (I1, I2). Of these forms, the L1 and L2 are associated with the membrane fraction, the E is predominantly secreted into the medium and the I1 and I2 are expressed as soluble proteins and surprisingly, a great portion accumulates in the culture medium. The molecular mass (94 kDa) of the expressed full-length receptor corresponds to the translation product of the entire cDNA coding region. The receptor biochemically identified in the rabbit mammary gland is however much shorter. Thus, in the mammary gland, the receptor presumably undergoes post-translational modifications. The receptor forms L1, L2 and S bind prolactin with specificity and affinity similar to those reported for the native receptor. They also interact with two monoclonal antibodies, M110 and A917, specific for the native conformation of the hormone-binding site. The I1 and I2 forms do not bind prolactin, whereas the E form does. Thus, the hormone binding site is located in the extracellular domain which can function autonomously as a PRL-binding soluble protein. However, the E form binds prolactin with a higher affinity than the native receptor and it does not bind one of the two antireceptor monoclonal antibodies, known to be hormone binding-site specific. Thus, the conformation of the native receptor and that of the E form differ.     

Integration of Motion Responses Underlying Directional Motion Anisotropy in Human Early Visual Cortical Areas

Recent imaging studies have reported directional motion biases in human visual cortex when perceiving moving random dot patterns. It has been hypothesized that these biases occur as a result of the integration of motion detector activation along the path of motion in visual cortex. In this study we investigate the nature of such motion integration with functional MRI (fMRI) using different motion stimuli. Three types of moving random dot stimuli were presented, showing either coherent motion, motion with spatial decorrelations or motion with temporal decorrelations. The results from the coherent motion stimulus reproduced the centripetal and centrifugal directional motion biases in V1, V2 and V3 as previously reported. The temporally decorrelated motion stimulus resulted in both centripetal and centrifugal biases similar to coherent motion. In contrast, the spatially decorrelated motion stimulus resulted in small directional motion biases that were only present in parts of visual cortex coding for higher eccentricities of the visual field. In combination with previous results, these findings indicate that biased motion responses in early visual cortical areas most likely depend on the spatial integration of a simultaneously activated motion detector chain.     

KCl evokes contraction of airway smooth muscle via activation of RhoA and Rho-kinase

Airway smooth muscle (ASM) cells express voltage-dependent Ca2+ channels, primarily of the L-subtype. These may play a role in excitation-contraction coupling of ASM, although other signaling pathways may also contribute: one of these includes Rho and its downstream effector molecule Rho-associated kinase (ROCK). Although voltage-dependent Ca2+ influx and Rho/ROCK signaling have traditionally been viewed as entirely separate pathways, recent evidence in vascular smooth muscle suggest differently. In this study, we monitored contractile activity (muscle baths) in bronchial and/or tracheal preparations from the pig, cow, and human, and further examined Rho and ROCK activities (Western blots and kinase assays) and cytosolic levels of Ca2+ (fluo 4-based fluorimetry) in porcine tracheal myocytes. KCl evoked substantial contractions that were suppressed in tracheal preparations by removal of external Ca2+ or using the selective L-type Ca2+ channel blocker nifedipine; porcine bronchial preparations were much less sensitive, and bovine bronchi were essentially unaffected by 1 microM nifedipine. Surprisingly, KCl-evoked contractions were also highly sensitive to two structurally different ROCK inhibitors: Y-27632 and HA-1077. Furthermore, the inhibitory effects of nifedipine and of the ROCK inhibitors were not additive. KCl also caused marked stimulation of Rho and ROCK activities, and both these changes were suppressed by nifedipine or by removal of external Ca2+. KCl-induced elevation of [Ca2+]i was not affected by Y-27632 but was reversed by NiCl2 or by BAPTA-AM. We conclude that KCl acts in part through stimulation of Rho and ROCK, possibly secondary to voltage-dependent Ca2+ influx.     

Acute changes in triacylglycerol lipase activity of human adipose tissue during exercise

Although physical exercise is known to increase adipose tissue lipolysis, its effect on the activity of triacylglycerol (TG) lipase, the enzyme regulating TG breakdown, is not known. The aim of the present study was to monitor the acute changes in TG lipase activity of adipose tissue induced during moderate exercise. For this purpose a new assay, sensitive to the phosphorylation state of the enzyme, was developed. Ten young sedentary men cycled for 30 min at a heart rate of 120-130 beats min(-1). Needle adipose tissue biopsy was performed from the buttock area at rest, at 5, 15, and 30 min of exercise, as well as at 15 min of passive recovery. Five other men served as controls by being biopsied as above without exercising. TG lipase activity was determined by measuring the decrease of endogenous TG concentration during incubation of the homogenized tissue. TG lipase activity increased 6.4-fold above baseline at 5 min of exercise (P < 0.001) and fell gradually afterwards, whereas it did not change significantly in the control group. In conclusion, our data show that TG lipase activity in human adipose tissue peaks early during exercise and subsequently decreases despite the maintenance of the physical stimulus.     

Novel selective thiazoleacetic acids as CRTH2 antagonists developed from in silico derived hits. Part 1

Structure–activity relationships of three related series of 4-phenylthiazol-5-ylacetic acids, derived from two hits emanating from a focused library obtained by in silico screening, have been explored as CRTH2 (chemoattractant receptor-homologous molecule expressed on Th2 cells) antagonists. Several compounds with double digit nanomolar binding affinity and full antagonistic efficacy for human CRTH2 receptor were obtained in all subclasses. The most potent compound was [2-(4-chloro-benzyl)-4-(4-phenoxy-phenyl)-thiazol-5-yl]acetic acid having an binding affinity of 3.7 nM and functional antagonistic effect of 66 nM in a BRET and 12 nM in a cAMP assay with no functional activity for the other PGD2 DP receptor (27 μM in cAMP).     

2‐D DIGE reveals changes in wheat xylanase inhibitor protein families due to Fusarium graminearum ΔTri5 infection and grain development

Wheat contains three different classes of proteinaceous xylanase inhibitors (XIs), i.e. Triticum aestivum xylanase inhibitors (TAXIs) xylanase‐inhibiting proteins (XIPs), and thaumatin‐like xylanase inhibitors (TLXIs) which are believed to act as a defensive barrier against phytopathogenic attack. In the absence of relevant data in wheat kernels, we here examined the response of the different members of the XI protein population to infection with a ΔTri5 mutant of Fusarium graminearum, the wild type of which is one of the most important wheat ear pathogens, in early developing wheat grain. Wheat ears were inoculated at anthesis, analyzed using 2‐D DIGE and multivariate analysis at 5, 15, and 25 days post anthesis (DPA), and compared with control samples. Distinct abundance patterns could be distinguished for different XI forms in response to infection with F. graminearum ΔTri5. Some (iso)forms were up‐regulated, whereas others were down‐regulated. This pathogen‐specific regulation of proteins was mostly visible at five DPA and levelled off in the samples situated further from the inoculation point. Furthermore, it was shown that most identified TAXI‐ and XIP‐type XI (iso)forms significantly increased in abundance from the milky (15 DPA) to the soft dough stages (25 DPA) on a per kernel basis, although the extent of increase differed greatly. Non‐glycosylated XIP forms increased more strongly than their glycosylated counterparts.     

The reverse mode of the Na(+)/Ca(2+) exchanger provides a source of Ca(2+) for store refilling following agonist-induced Ca(2+) mobilization

Agonist-induced contraction of airway smooth muscle (ASM) can be triggered by an elevation in the intracellular Ca(2+) concentration, primarily through the release of Ca(2+) from the sarcoplasmic reticulum (SR). The refilling of the SR is integral for subsequent contractions. It has been suggested that Ca(2+) entry via store-operated cation (SOC) and receptor-operated cation channels may facilitate refilling of the SR. Indeed, depletion of the SR activates substantial inward SOC currents in ASM that are composed of both Ca(2+) and Na(+). Accumulation of Na(+) within the cell may regulate Ca(2+) handling in ASM by forcing the Na(+)/Ca(2+) exchanger (NCX) into the reverse mode, leading to the influx of Ca(2+) from the extracellular domain. Since depletion of the SR activates substantial inward Na(+) current, it is conceivable that the reverse mode of the NCX may contribute to the intracellular Ca(2+) pool from which the SR is refilled. Indeed, successive contractions of bovine ASM, evoked by various agonists (ACh, histamine, 5-HT, caffeine) were significantly reduced upon removal of extracellular Na(+); whereas contractions evoked by KCl were unchanged by Na(+) depletion. Ouabain, a selective inhibitor of the Na(+)/K(+) pump, had no effect on the reductions observed under normal and zero-Na(+) conditions. KB-R7943, a selective inhibitor of the reverse mode of the NCX, significantly reduced successive contractions induced by all agonists without altering KCl responses. Furthermore, KB-R7943 abolished successive caffeine-induced Ca(2+) transients in single ASM cells. Together, these data suggest a role for the reverse mode of the NCX in refilling the SR in ASM following Ca(2+) mobilization.